Publications by authors named "Stuart H Orkin"

301 Publications

Dietary suppression of MHC class II expression in intestinal epithelial cells enhances intestinal tumorigenesis.

Cell Stem Cell 2021 Sep 15. Epub 2021 Sep 15.

The David H. Koch Institute for Integrative Cancer Research at MIT, Department of Biology, MIT, Cambridge, MA 02139, USA; Klarman Cell Observatory, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02140, USA.

Little is known about how interactions of diet, intestinal stem cells (ISCs), and immune cells affect early-stage intestinal tumorigenesis. We show that a high-fat diet (HFD) reduces the expression of the major histocompatibility complex class II (MHC class II) genes in intestinal epithelial cells, including ISCs. This decline in epithelial MHC class II expression in a HFD correlates with reduced intestinal microbiome diversity. Microbial community transfer experiments suggest that epithelial MHC class II expression is regulated by intestinal flora. Mechanistically, pattern recognition receptor (PRR) and interferon-gamma (IFNγ) signaling regulates epithelial MHC class II expression. MHC class II-negative (MHC-II-) ISCs exhibit greater tumor-initiating capacity than their MHC class II-positive (MHC-II+) counterparts upon loss of the tumor suppressor Apc coupled with a HFD, suggesting a role for epithelial MHC class II-mediated immune surveillance in suppressing tumorigenesis. ISC-specific genetic ablation of MHC class II increases tumor burden cell autonomously. Thus, HFD perturbs a microbiome-stem cell-immune cell interaction that contributes to tumor initiation in the intestine.
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http://dx.doi.org/10.1016/j.stem.2021.08.007DOI Listing
September 2021

Mapping the evolving landscape of super-enhancers during cell differentiation.

Genome Biol 2021 09 15;22(1):269. Epub 2021 Sep 15.

Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, 02115, USA.

Background: Super-enhancers are clusters of enhancer elements that play critical roles in the maintenance of cell identity. Current investigations on super-enhancers are centered on the established ones in static cell types. How super-enhancers are established during cell differentiation remains obscure.

Results: Here, by developing an unbiased approach to systematically analyze the evolving landscape of super-enhancers during cell differentiation in multiple lineages, we discover a general trend where super-enhancers emerge through three distinct temporal patterns: conserved, temporally hierarchical, and de novo. The three types of super-enhancers differ further in association patterns in target gene expression, functional enrichment, and 3D chromatin organization, suggesting they may represent distinct structural and functional subtypes. Furthermore, we dissect the enhancer repertoire within temporally hierarchical super-enhancers, and find enhancers that emerge at early and late stages are enriched with distinct transcription factors, suggesting that the temporal order of establishment of elements within super-enhancers may be directed by underlying DNA sequence. CRISPR-mediated deletion of individual enhancers in differentiated cells shows that both the early- and late-emerged enhancers are indispensable for target gene expression, while in undifferentiated cells early enhancers are involved in the regulation of target genes.

Conclusions: In summary, our analysis highlights the heterogeneity of the super-enhancer population and provides new insights to enhancer functions within super-enhancers.
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http://dx.doi.org/10.1186/s13059-021-02485-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8442463PMC
September 2021

A unified model of human hemoglobin switching through single-cell genome editing.

Nat Commun 2021 08 17;12(1):4991. Epub 2021 Aug 17.

Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.

Key mechanisms of fetal hemoglobin (HbF) regulation and switching have been elucidated through studies of human genetic variation, including mutations in the HBG1/2 promoters, deletions in the β-globin locus, and variation impacting BCL11A. While this has led to substantial insights, there has not been a unified understanding of how these distinct genetically-nominated elements, as well as other key transcription factors such as ZBTB7A, collectively interact to regulate HbF. A key limitation has been the inability to model specific genetic changes in primary isogenic human hematopoietic cells to uncover how each of these act individually and in aggregate. Here, we describe a single-cell genome editing functional assay that enables specific mutations to be recapitulated individually and in combination, providing insights into how multiple mutation-harboring functional elements collectively contribute to HbF expression. In conjunction with quantitative modeling and chromatin capture analyses, we illustrate how these genetic findings enable a comprehensive understanding of how distinct regulatory mechanisms can synergistically modulate HbF expression.
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http://dx.doi.org/10.1038/s41467-021-25298-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8371164PMC
August 2021

Reactivation of a developmentally silenced embryonic globin gene.

Nat Commun 2021 07 21;12(1):4439. Epub 2021 Jul 21.

MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

The α- and β-globin loci harbor developmentally expressed genes, which are silenced throughout post-natal life. Reactivation of these genes may offer therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address mechanisms regulating the embryonically expressed α-like globin, termed ζ-globin. We show that in embryonic erythroid cells, the ζ-gene lies within a ~65 kb sub-TAD (topologically associating domain) of open, acetylated chromatin and interacts with the α-globin super-enhancer. By contrast, in adult erythroid cells, the ζ-gene is packaged within a small (~10 kb) sub-domain of hypoacetylated, facultative heterochromatin within the acetylated sub-TAD and that it no longer interacts with its enhancers. The ζ-gene can be partially re-activated by acetylation and inhibition of histone de-acetylases. In addition to suggesting therapies for severe α-thalassemia, these findings illustrate the general principles by which reactivation of developmental genes may rescue abnormalities arising from mutations in their adult paralogues.
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http://dx.doi.org/10.1038/s41467-021-24402-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8295333PMC
July 2021

Indispensable epigenetic control of thymic epithelial cell development and function by polycomb repressive complex 2.

Nat Commun 2021 06 24;12(1):3933. Epub 2021 Jun 24.

Department of Biomedicine and University Children's Hospital of Basel, University of Basel, Basel, Switzerland.

Thymic T cell development and T cell receptor repertoire selection are dependent on essential molecular cues provided by thymic epithelial cells (TEC). TEC development and function are regulated by their epigenetic landscape, in which the repressive H3K27me3 epigenetic marks are catalyzed by polycomb repressive complex 2 (PRC2). Here we show that a TEC-targeted deficiency of PRC2 function results in a hypoplastic thymus with reduced ability to express antigens and select a normal repertoire of T cells. The absence of PRC2 activity reveals a transcriptomically distinct medullary TEC lineage that incompletely off-sets the shortage of canonically-derived medullary TEC whereas cortical TEC numbers remain unchanged. This alternative TEC development is associated with the generation of reduced TCR diversity. Hence, normal PRC2 activity and placement of H3K27me3 marks are required for TEC lineage differentiation and function and, in their absence, the thymus is unable to compensate for the loss of a normal TEC scaffold.
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http://dx.doi.org/10.1038/s41467-021-24158-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8225857PMC
June 2021

Author Correction: Transcription factor competition at the γ-globin promoters controls hemoglobin switching.

Nat Genet 2021 Apr;53(4):586

Cancer and Blood Disorders Center, Dana-Farber Cancer Institute and Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.

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http://dx.doi.org/10.1038/s41588-021-00834-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8189157PMC
April 2021

MOLECULAR MEDICINE: Found in Translation.

Authors:
Stuart H Orkin

Med (N Y) 2021 Feb 12;2(2):122-136. Epub 2021 Jan 12.

Dana Farber/Boston Children's Cancer & Blood Disorders Center, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115.

Studies of the major hemoglobin disorders, β-thalassemia and sickle cell disease (SCD), have laid a foundation for molecular medicine. While enormous progress has been made in understanding gene structure and regulation, translating molecular insights to therapy for the many individuals affected with these disorders has been challenging. Advances in three activities have recently converged to bring novel genetic and potentially curative treatments to clinical trials. First, improved lentiviral vectors for gene transfer into hematopoietic stem cells have revived somatic gene therapy for blood disorders. Second, elucidation of regulatory factors and mechanisms that control the normal developmental switch from fetal to adult hemoglobin has provided a route to reactivation of the fetal form for therapy. Third, revolutionary methods of gene engineering permit molecular insights to be leveraged for patients. Here I review how the promise of molecular medicine to bring transformative treatments to the clinical arena is finally being realized.
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http://dx.doi.org/10.1016/j.medj.2020.12.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939134PMC
February 2021

Transcription factor competition at the γ-globin promoters controls hemoglobin switching.

Nat Genet 2021 04 1;53(4):511-520. Epub 2021 Mar 1.

Cancer and Blood Disorders Center, Dana-Farber Cancer Institute and Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.

BCL11A, the major regulator of fetal hemoglobin (HbF, αγ) level, represses γ-globin expression through direct promoter binding in adult erythroid cells in a switch to adult hemoglobin (HbA, αβ). To uncover how BCL11A initiates repression, we used CRISPR-Cas9, dCas9, dCas9-KRAB and dCas9-VP64 screens to dissect the γ-globin promoters and identified an activator element near the BCL11A-binding site. Using CUT&RUN and base editing, we demonstrate that a proximal CCAAT box is occupied by the activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate repression. Occupancy of NF-Y is rapidly established following BCL11A depletion, and precedes γ-globin derepression and locus control region (LCR)-globin loop formation. Our findings reveal that the switch from fetal to adult globin gene expression within the >50-kb β-globin gene cluster is initiated by competition between a stage-selective repressor and a ubiquitous activating factor within a remarkably discrete region of the γ-globin promoters.
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http://dx.doi.org/10.1038/s41588-021-00798-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038971PMC
April 2021

ARID4B is critical for mouse embryonic stem cell differentiation towards mesoderm and endoderm, linking epigenetics to pluripotency exit.

J Biol Chem 2020 12;295(51):17738-17751

Howard Hughes Medical Institute, Dana Farber/Boston Children's Cancer and Blood Disorders Center, Dept. of Pediatrics, Harvard Medical School, Boston, Massachusetts USA. Electronic address:

Distinct cell types emerge from embryonic stem cells through a precise and coordinated execution of gene expression programs during lineage commitment. This is established by the action of lineage specific transcription factors along with chromatin complexes. Numerous studies have focused on epigenetic factors that affect embryonic stem cells (ESC) self-renewal and pluripotency. However, the contribution of chromatin to lineage decisions at the exit from pluripotency has not been as extensively studied. Using a pooled epigenetic shRNA screen strategy, we identified chromatin-related factors critical for differentiation toward mesodermal and endodermal lineages. Here we reveal a critical role for the chromatin protein, ARID4B. Arid4b-deficient mESCs are similar to WT mESCs in the expression of pluripotency factors and their self-renewal. However, ARID4B loss results in defects in up-regulation of the meso/endodermal gene expression program. It was previously shown that Arid4b resides in a complex with SIN3A and HDACS 1 and 2. We identified a physical and functional interaction of ARID4B with HDAC1 rather than HDAC2, suggesting functionally distinct Sin3a subcomplexes might regulate cell fate decisions Finally, we observed that ARID4B deficiency leads to increased H3K27me3 and a reduced H3K27Ac level in key developmental gene loci, whereas a subset of genomic regions gain H3K27Ac marks. Our results demonstrate that epigenetic control through ARID4B plays a key role in the execution of lineage-specific gene expression programs at pluripotency exit.
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http://dx.doi.org/10.1074/jbc.RA120.015534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762958PMC
December 2020

A saturating mutagenesis CRISPR-Cas9-mediated functional genomic screen identifies and regulatory elements of in murine ESCs.

J Biol Chem 2020 11 29;295(47):15797-15809. Epub 2020 Sep 29.

Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia; Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia. Electronic address:

Regulatory elements (REs) consist of enhancers and promoters that occupy a significant portion of the noncoding genome and control gene expression programs either in or in Putative REs have been identified largely based on their regulatory features (co-occupancy of ESC-specific transcription factors, enhancer histone marks, and DNase hypersensitivity) in mouse embryonic stem cells (mESCs). However, less has been established regarding their regulatory functions in their native context. We deployed and regulatory elements scanning through saturating mutagenesis and sequencing (ctSCAN-SMS) to target elements within the ∼12-kb -region (REs; CREs) of the gene locus, as well as genome-wide 2,613 high-confidence REs (TREs), in mESCs. ctSCAN-SMS identified 10 CREs and 12 TREs as novel candidate REs of the gene in mESCs. Furthermore, deletions of these candidate REs confirmed that the majority of the REs are functionally active, and CREs are more active than TREs in controlling gene expression. A subset of active CREs and TREs physically interact with the promoter to varying degrees; specifically, a greater number of active CREs, compared with active TREs, physically interact with the promoter. Moreover, comparative genomics analysis reveals that a greater number of active CREs than active TREs are evolutionarily conserved between mice and primates, including humans. Taken together, our study demonstrates the reliability and robustness of ctSCAN-SMS screening to identify critical REs and investigate their roles in the regulation of transcriptional output of a target gene (in this case ) in their native context.
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http://dx.doi.org/10.1074/jbc.RA120.013772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7681025PMC
November 2020

Enhancer dependence of cell-type-specific gene expression increases with developmental age.

Proc Natl Acad Sci U S A 2020 09 19;117(35):21450-21458. Epub 2020 Aug 19.

Cancer and Blood Disorders Center, Boston Children's Hospital and Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215;

How overall principles of cell-type-specific gene regulation (the "logic") may change during ontogeny is largely unexplored. We compared transcriptomic, epigenomic, and three-dimensional (3D) genomic profiles in embryonic (EryP) and adult (EryD) erythroblasts. Despite reduced chromatin accessibility compared to EryP, distal chromatin of EryD is enriched in H3K27ac, Gata1, and Myb occupancy. EryP-/EryD-shared enhancers are highly correlated with red blood cell identity genes, whereas cell-type-specific regulation employs different elements in EryP and EryD cells. In contrast to EryP-specific genes, which exhibit promoter-centric regulation through Gata1, EryD-specific genes rely more on distal enhancers for regulation involving Myb-mediated enhancer activation. Gata1 HiChIP demonstrated an overall increased enhancer-promoter interactions at EryD-specific genes, whereas genome editing in selected loci confirmed distal enhancers are required for gene expression in EryD but not in EryP. Applying a metric for enhancer dependence of transcription, we observed a progressive reliance on cell-specific enhancers with increasing ontogenetic age among diverse tissues of mouse and human origin. Our findings highlight fundamental and conserved differences at distinct developmental stages, characterized by simpler promoter-centric regulation of cell-type-specific genes in embryonic cells and increased combinatorial enhancer-driven control in adult cells.
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http://dx.doi.org/10.1073/pnas.2008672117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474592PMC
September 2020

An Engineered CRISPR-Cas9 Mouse Line for Simultaneous Readout of Lineage Histories and Gene Expression Profiles in Single Cells.

Cell 2020 06 14;181(6):1410-1422.e27. Epub 2020 May 14.

Stem Cell Program, Boston Children's Hospital, Boston, MA, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA. Electronic address:

Tracing the lineage history of cells is key to answering diverse and fundamental questions in biology. Coupling of cell ancestry information with other molecular readouts represents an important goal in the field. Here, we describe the CRISPR array repair lineage tracing (CARLIN) mouse line and corresponding analysis tools that can be used to simultaneously interrogate the lineage and transcriptomic information of single cells in vivo. This model exploits CRISPR technology to generate up to 44,000 transcribed barcodes in an inducible fashion at any point during development or adulthood, is compatible with sequential barcoding, and is fully genetically defined. We have used CARLIN to identify intrinsic biases in the activity of fetal liver hematopoietic stem cell (HSC) clones and to uncover a previously unappreciated clonal bottleneck in the response of HSCs to injury. CARLIN also allows the unbiased identification of transcriptional signatures associated with HSC activity without cell sorting.
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http://dx.doi.org/10.1016/j.cell.2020.04.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529102PMC
June 2020

Multiplexed capture of spatial configuration and temporal dynamics of locus-specific 3D chromatin by biotinylated dCas9.

Genome Biol 2020 03 5;21(1):59. Epub 2020 Mar 5.

Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.

The spatiotemporal control of 3D genome is fundamental for gene regulation, yet it remains challenging to profile high-resolution chromatin structure at cis-regulatory elements (CREs). Using C-terminally biotinylated dCas9, endogenous biotin ligases, and pooled sgRNAs, we describe the dCas9-based CAPTURE method for multiplexed analysis of locus-specific chromatin interactions. The redesigned system allows for quantitative analysis of the spatial configuration of a few to hundreds of enhancers or promoters in a single experiment, enabling comparisons across CREs within and between gene clusters. Multiplexed analyses of the spatiotemporal configuration of erythroid super-enhancers and promoter-centric interactions reveal organizational principles of genome structure and function.
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http://dx.doi.org/10.1186/s13059-020-01973-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059722PMC
March 2020

Live-animal imaging of native haematopoietic stem and progenitor cells.

Nature 2020 02 5;578(7794):278-283. Epub 2020 Feb 5.

Stem Cell Program, Boston Children's Hospital, Boston, MA, USA.

The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions. It has been particularly challenging to study dynamic HSC behaviour, given that the visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow. We show that this subset of LT-HSCs resides close to both sinusoidal blood vessels and the endosteal surface. By contrast, multipotent progenitor cells (MPPs) show greater variation in distance from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in bone marrow niches with the deepest hypoxia and instead are found in hypoxic environments similar to those of MPPs. In vivo time-lapse imaging revealed that LT-HSCs at steady-state show limited motility. Activated LT-HSCs show heterogeneous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities with bone-remodelling activity. By contrast, cavities with low bone-resorbing activity do not harbour expanding HSCs. These findings point to previously unknown heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover. Our approach enables the direct visualization of HSC behaviours and dissection of heterogeneity in HSC niches.
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http://dx.doi.org/10.1038/s41586-020-1971-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021587PMC
February 2020

Control of human hemoglobin switching by LIN28B-mediated regulation of BCL11A translation.

Nat Genet 2020 02 20;52(2):138-145. Epub 2020 Jan 20.

Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.

Increased production of fetal hemoglobin (HbF) can ameliorate the severity of sickle cell disease and β-thalassemia. BCL11A represses the genes encoding HbF and regulates human hemoglobin switching through variation in its expression during development. However, the mechanisms underlying the developmental expression of BCL11A remain mysterious. Here we show that BCL11A is regulated at the level of messenger RNA (mRNA) translation during human hematopoietic development. Despite decreased BCL11A protein synthesis earlier in development, BCL11A mRNA continues to be associated with ribosomes. Through unbiased genomic and proteomic analyses, we demonstrate that the RNA-binding protein LIN28B, which is developmentally expressed in a pattern reciprocal to that of BCL11A, directly interacts with ribosomes and BCL11A mRNA. Furthermore, we show that BCL11A mRNA translation is suppressed by LIN28B through direct interactions, independently of its role in regulating let-7 microRNAs, and that BCL11A is the major target of LIN28B-mediated HbF induction. Our results reveal a previously unappreciated mechanism underlying human hemoglobin switching that illuminates new therapeutic opportunities.
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http://dx.doi.org/10.1038/s41588-019-0568-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031047PMC
February 2020

CUT&RUNTools: a flexible pipeline for CUT&RUN processing and footprint analysis.

Genome Biol 2019 09 9;20(1):192. Epub 2019 Sep 9.

Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard Chan School of Public Health, Boston, MA, USA.

We introduce CUT&RUNTools as a flexible, general pipeline for facilitating the identification of chromatin-associated protein binding and genomic footprinting analysis from antibody-targeted CUT&RUN primary cleavage data. CUT&RUNTools extracts endonuclease cut site information from sequences of short-read fragments and produces single-locus binding estimates, aggregate motif footprints, and informative visualizations to support the high-resolution mapping capability of CUT&RUN. CUT&RUNTools is available at https://bitbucket.org/qzhudfci/cutruntools/ .
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http://dx.doi.org/10.1186/s13059-019-1802-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6734249PMC
September 2019

BORIS promotes chromatin regulatory interactions in treatment-resistant cancer cells.

Nature 2019 08 7;572(7771):676-680. Epub 2019 Aug 7.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

The CCCTC-binding factor (CTCF), which anchors DNA loops that organize the genome into structural domains, has a central role in gene control by facilitating or constraining interactions between genes and their regulatory elements. In cancer cells, the disruption of CTCF binding at specific loci by somatic mutation or DNA hypermethylation results in the loss of loop anchors and consequent activation of oncogenes. By contrast, the germ-cell-specific paralogue of CTCF, BORIS (brother of the regulator of imprinted sites, also known as CTCFL), is overexpressed in several cancers, but its contributions to the malignant phenotype remain unclear. Here we show that aberrant upregulation of BORIS promotes chromatin interactions in ALK-mutated, MYCN-amplified neuroblastoma cells that develop resistance to ALK inhibition. These cells are reprogrammed to a distinct phenotypic state during the acquisition of resistance, a process defined by the initial loss of MYCN expression followed by subsequent overexpression of BORIS and a concomitant switch in cellular dependence from MYCN to BORIS. The resultant BORIS-regulated alterations in chromatin looping lead to the formation of super-enhancers that drive the ectopic expression of a subset of proneural transcription factors that ultimately define the resistance phenotype. These results identify a previously unrecognized role of BORIS-to promote regulatory chromatin interactions that support specific cancer phenotypes.
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http://dx.doi.org/10.1038/s41586-019-1472-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010522PMC
August 2019

Rational targeting of a NuRD subcomplex guided by comprehensive in situ mutagenesis.

Nat Genet 2019 07 28;51(7):1149-1159. Epub 2019 Jun 28.

Division of Hematology/Oncology, Boston Children's Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Broad Institute, Harvard Medical School, Boston, MA, USA.

Developmental silencing of fetal globins serves as both a paradigm of spatiotemporal gene regulation and an opportunity for therapeutic intervention of β-hemoglobinopathy. The nucleosome remodeling and deacetylase (NuRD) chromatin complex participates in γ-globin repression. We used pooled CRISPR screening to disrupt NuRD protein coding sequences comprehensively in human adult erythroid precursors. Essential for fetal hemoglobin (HbF) control is a non-redundant subcomplex of NuRD protein family paralogs, whose composition we corroborated by affinity chromatography and proximity labeling mass spectrometry proteomics. Mapping top functional guide RNAs identified key protein interfaces where in-frame alleles resulted in loss-of-function due to destabilization or altered function of subunits. We ascertained mutations of CHD4 that dissociate its requirement for cell fitness from HbF repression in both primary human erythroid precursors and transgenic mice. Finally we demonstrated that sequestering CHD4 from NuRD phenocopied these mutations. These results indicate a generalizable approach to discover protein complex features amenable to rational biochemical targeting.
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http://dx.doi.org/10.1038/s41588-019-0453-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650275PMC
July 2019

TAF5L and TAF6L Maintain Self-Renewal of Embryonic Stem Cells via the MYC Regulatory Network.

Mol Cell 2019 06 17;74(6):1148-1163.e7. Epub 2019 Apr 17.

Department of Anatomy and Developmental Biology, Monash University, Wellington Road, Clayton, VIC 3800, Australia; Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Wellington Road, Clayton, VIC 3800, Australia. Electronic address:

Self-renewal and pluripotency of the embryonic stem cell (ESC) state are established and maintained by multiple regulatory networks that comprise transcription factors and epigenetic regulators. While much has been learned regarding transcription factors, the function of epigenetic regulators in these networks is less well defined. We conducted a CRISPR-Cas9-mediated loss-of-function genetic screen that identified two epigenetic regulators, TAF5L and TAF6L, components or co-activators of the GNAT-HAT complexes for the mouse ESC (mESC) state. Detailed molecular studies demonstrate that TAF5L/TAF6L transcriptionally activate c-Myc and Oct4 and their corresponding MYC and CORE regulatory networks. Besides, TAF5L/TAF6L predominantly regulate their target genes through H3K9ac deposition and c-MYC recruitment that eventually activate the MYC regulatory network for self-renewal of mESCs. Thus, our findings uncover a role of TAF5L/TAF6L in directing the MYC regulatory network that orchestrates gene expression programs to control self-renewal for the maintenance of mESC state.
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http://dx.doi.org/10.1016/j.molcel.2019.03.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6671628PMC
June 2019

Extensive Recovery of Embryonic Enhancer and Gene Memory Stored in Hypomethylated Enhancer DNA.

Mol Cell 2019 05 21;74(3):542-554.e5. Epub 2019 Mar 21.

Department of Medical Oncology and Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Medicine, Brigham & Women's Hospital, Boston, MA 02215, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA. Electronic address:

Developing and adult tissues use different cis-regulatory elements. Although DNA at some decommissioned embryonic enhancers is hypomethylated in adult cells, it is unknown whether this putative epigenetic memory is complete and recoverable. We find that, in adult mouse cells, hypomethylated CpG dinucleotides preserve a nearly complete archive of tissue-specific developmental enhancers. Sites that carry the active histone mark H3K4me1, and are therefore considered "primed," are mainly cis elements that act late in organogenesis. In contrast, sites decommissioned early in development retain hypomethylated DNA as a singular property. In adult intestinal and blood cells, sustained absence of polycomb repressive complex 2 indirectly reactivates most-and only-hypomethylated developmental enhancers. Embryonic and fetal transcriptional programs re-emerge as a result, in reverse chronology to cis element inactivation during development. Thus, hypomethylated DNA in adult cells preserves a "fossil record" of tissue-specific developmental enhancers, stably marking decommissioned sites and enabling recovery of this epigenetic memory.
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http://dx.doi.org/10.1016/j.molcel.2019.02.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6499659PMC
May 2019

Single-Cell Analysis Identifies LY6D as a Marker Linking Castration-Resistant Prostate Luminal Cells to Prostate Progenitors and Cancer.

Cell Rep 2018 12;25(12):3504-3518.e6

Prostate Oncobiology, Cancer Research UK Manchester Institute, The University of Manchester, Alderley Park SK10 4TG, UK; Belfast-Manchester Movember Centre of Excellence, Cancer Research UK Manchester Institute, The University of Manchester, Alderley Park SK10 4TG, UK. Electronic address:

The exact identity of castrate-resistant (CR) cells and their relation to CR prostate cancer (CRPC) is unresolved. We use single-cell gene profiling to analyze the molecular heterogeneity in basal and luminal compartments. Within the luminal compartment, we identify a subset of cells intrinsically resistant to castration with a bi-lineage gene expression pattern. We discover LY6D as a marker of CR prostate progenitors with multipotent differentiation and enriched organoid-forming capacity. Lineage tracing further reveals that LY6D CR luminal cells can produce LY6D luminal cells. In contrast, in luminal cells lacking PTEN, LY6D cells predominantly give rise to LY6D tumor cells, contributing to high-grade PIN lesions. Gene expression analyses in patients' biopsies indicate that LY6D expression correlates with early disease progression, including progression to CRPC. Our studies thus identify a subpopulation of luminal progenitors characterized by LY6D expression and intrinsic castration resistance. LY6D may serve as a prognostic maker for advanced prostate cancer.
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http://dx.doi.org/10.1016/j.celrep.2018.11.069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315111PMC
December 2018

Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation.

Elife 2018 12 18;7. Epub 2018 Dec 18.

Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States.

Approximately, 30% of embryonic stem cells (ESCs) die after exiting self-renewal, but regulators of this process are not well known. Yap1 is a Hippo pathway transcriptional effector that plays numerous roles in development and cancer. However, its functions in ESC differentiation remain poorly characterized. We first reveal that ESCs lacking Yap1 experience massive cell death upon the exit from self-renewal. We subsequently show that Yap1 contextually protects differentiating, but not self-renewing, ESC from hyperactivation of the apoptotic cascade. Mechanistically, Yap1 strongly activates anti-apoptotic genes via regulatory elements while mildly suppressing pro-apoptotic genes, which moderates the level of mitochondrial priming that occurs during differentiation. Individually modulating the expression of single apoptosis-related genes targeted by Yap1 is sufficient to augment or hinder survival during differentiation. Our demonstration of the context-dependent pro-survival functions of Yap1 during ESC differentiation contributes to our understanding of the balance between survival and death during cell fate changes.
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http://dx.doi.org/10.7554/eLife.40167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307859PMC
December 2018

PRC2 loss induces chemoresistance by repressing apoptosis in T cell acute lymphoblastic leukemia.

J Exp Med 2018 12 7;215(12):3094-3114. Epub 2018 Nov 7.

Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA

The tendency of mitochondria to undergo or resist BCL2-controlled apoptosis (so-called mitochondrial priming) is a powerful predictor of response to cytotoxic chemotherapy. Fully exploiting this finding will require unraveling the molecular genetics underlying phenotypic variability in mitochondrial priming. Here, we report that mitochondrial apoptosis resistance in T cell acute lymphoblastic leukemia (T-ALL) is mediated by inactivation of polycomb repressive complex 2 (PRC2). In T-ALL clinical specimens, loss-of-function mutations of PRC2 core components (, , or ) were associated with mitochondrial apoptosis resistance. In T-ALL cells, PRC2 depletion induced resistance to apoptosis induction by multiple chemotherapeutics with distinct mechanisms of action. PRC2 loss induced apoptosis resistance via transcriptional up-regulation of the LIM domain transcription factor and downstream up-regulation of the mitochondrial chaperone These findings demonstrate the importance of mitochondrial apoptotic priming as a prognostic factor in T-ALL and implicate mitochondrial chaperone function as a molecular determinant of chemotherapy response.
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http://dx.doi.org/10.1084/jem.20180570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279404PMC
December 2018

FAM210B is an erythropoietin target and regulates erythroid heme synthesis by controlling mitochondrial iron import and ferrochelatase activity.

J Biol Chem 2018 12 26;293(51):19797-19811. Epub 2018 Oct 26.

the Division of Hematology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115.

Erythropoietin (EPO) signaling is critical to many processes essential to terminal erythropoiesis. Despite the centrality of iron metabolism to erythropoiesis, the mechanisms by which EPO regulates iron status are not well-understood. To this end, here we profiled gene expression in EPO-treated 32D pro-B cells and developing fetal liver erythroid cells to identify additional iron regulatory genes. We determined that FAM210B, a mitochondrial inner-membrane protein, is essential for hemoglobinization, proliferation, and enucleation during terminal erythroid maturation. deficiency led to defects in mitochondrial iron uptake, heme synthesis, and iron-sulfur cluster formation. These defects were corrected with a lipid-soluble, small-molecule iron transporter, hinokitiol, in -deficient murine erythroid cells and zebrafish morphants. Genetic complementation experiments revealed that FAM210B is not a mitochondrial iron transporter but is required for adequate mitochondrial iron import to sustain heme synthesis and iron-sulfur cluster formation during erythroid differentiation. FAM210B was also required for maximal ferrochelatase activity in differentiating erythroid cells. We propose that FAM210B functions as an adaptor protein that facilitates the formation of an oligomeric mitochondrial iron transport complex, required for the increase in iron acquisition for heme synthesis during terminal erythropoiesis. Collectively, our results reveal a critical mechanism by which EPO signaling regulates terminal erythropoiesis and iron metabolism.
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http://dx.doi.org/10.1074/jbc.RA118.002742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314115PMC
December 2018

Emerging Genetic Therapy for Sickle Cell Disease.

Annu Rev Med 2019 01 24;70:257-271. Epub 2018 Oct 24.

Dana Farber/Boston Children's Cancer and Blood Disorders Center, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA; email:

The genetic basis of sickle cell disease (SCD) was elucidated >60 years ago, yet current therapy does not rely on this knowledge. Recent advances raise prospects for improved, and perhaps curative, treatment. First, transcription factors, BCL11A and LRF/ZBTB7A, that mediate silencing of the β-like fetal (γ-) globin gene after birth have been identified and demonstrated to act at the γ-globin promoters, precisely at recognition sequences disrupted in rare individuals with hereditary persistence of fetal hemoglobin. Second, transformative advances in gene editing and progress in lentiviral gene therapy provide diverse opportunities for genetic strategies to cure SCD. Approaches include hematopoietic gene therapy by globin gene addition, gene editing to correct the SCD mutation, and genetic manipulations to enhance fetal hemoglobin production, a potent modifier of the clinical phenotype. Clinical trials may soon identify efficacious and safe genetic approaches to the ultimate goal of cure for SCD.
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http://dx.doi.org/10.1146/annurev-med-041817-125507DOI Listing
January 2019

Polycomb Repressive Complex 2 is essential for development and maintenance of a functional TEC compartment.

Sci Rep 2018 09 25;8(1):14335. Epub 2018 Sep 25.

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Science Park, Smithville, Texas, 78957, USA.

Thymic epithelial cells (TEC) are essential for thymocyte differentiation and repertoire selection. Despite their indispensable role in generating functional T cells, the molecular mechanisms that orchestrate TEC development from endodermal progenitors in the third pharyngeal pouch (3 PP) are not fully understood. We recently reported that the T-box transcription factor TBX1 negatively regulates TEC development. Although initially expressed throughout the 3 PP, Tbx1 becomes downregulated in thymus-fated progenitors and when ectopically expressed impairs TEC progenitor proliferation and differentiation. Here we show that ectopic Tbx1 expression in thymus fated endoderm increases expression of Polycomb repressive complex 2 (PRC2) target genes in TEC. PRC2 is an epigenetic modifier that represses gene expression by catalyzing trimethylation of lysine 27 on histone H3. The increased expression of PRC2 target genes suggests that ectopic Tbx1 interferes with PRC2 activity and implicates PRC2 as an important regulator of TEC development. To test this hypothesis, we used Foxn1 to delete Eed, a PRC2 component required for complex stability and function in thymus fated 3 PP endoderm. Proliferation and differentiation of fetal and newborn TEC were disrupted in the conditional knockout (Eed) mutants leading to severely dysplastic adult thymi. Consistent with PRC2-mediated transcriptional silencing, the majority of differentially expressed genes (DEG) were upregulated in Eed TEC. Moreover, a high frequency of Eed DEG overlapped with DEG in TEC that ectopically expressed Tbx1. These findings demonstrate that PRC2 plays a critical role in TEC development and suggest that Tbx1 expression must be downregulated in thymus fated 3 PP endoderm to ensure optimal PRC2 function.
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http://dx.doi.org/10.1038/s41598-018-32729-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156232PMC
September 2018

Canonical PRC2 function is essential for mammary gland development and affects chromatin compaction in mammary organoids.

PLoS Biol 2018 08 6;16(8):e2004986. Epub 2018 Aug 6.

ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

Distinct transcriptional states are maintained through organization of chromatin, resulting from the sum of numerous repressive and active histone modifications, into tightly packaged heterochromatin versus more accessible euchromatin. Polycomb repressive complex 2 (PRC2) is the main mammalian complex responsible for histone 3 lysine 27 trimethylation (H3K27me3) and is integral to chromatin organization. Using in vitro and in vivo studies, we show that deletion of Suz12, a core component of all PRC2 complexes, results in loss of H3K27me3 and H3K27 dimethylation (H3K27me2), completely blocks normal mammary gland development, and profoundly curtails progenitor activity in 3D organoid cultures. Through the application of mammary organoids to bypass the severe phenotype associated with Suz12 loss in vivo, we have explored gene expression and chromatin structure in wild-type and Suz12-deleted basal-derived organoids. Analysis of organoids led to the identification of lineage-specific changes in gene expression and chromatin structure, inferring cell type-specific PRC2-mediated gene silencing of the chromatin state. These expression changes were accompanied by cell cycle arrest but not lineage infidelity. Together, these data indicate that canonical PRC2 function is essential for development of the mammary gland through the repression of alternate transcription programs and maintenance of chromatin states.
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http://dx.doi.org/10.1371/journal.pbio.2004986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095611PMC
August 2018

Author Correction: High-fat diet enhances stemness and tumorigenicity of intestinal progenitors.

Nature 2018 08;560(7717):E26

The David H. Koch Institute for Integrative Cancer Research at MIT, Department of Biology, MIT, Cambridge, Massachusetts, 02139, USA.

In Fig. 4e of this Article, the labels for 'Control' and 'HFD' were reversed ('Control' should have been labelled blue rather than purple, and 'HFD' should have been labelled purple rather than blue). Similarly, in Fig. 4f of this Article, the labels for 'V' and 'GW' were reversed ('V' should have been labelled blue rather than purple, and 'GW' should have been labelled purple instead of blue). The original figure has been corrected online.
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http://dx.doi.org/10.1038/s41586-018-0187-yDOI Listing
August 2018
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