Publications by authors named "Satish K Nandakumar"

13 Publications

  • Page 1 of 1

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

Common variants in signaling transcription-factor-binding sites drive phenotypic variability in red blood cell traits.

Nat Genet 2020 12 23;52(12):1333-1345. Epub 2020 Nov 23.

Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

Genome-wide association studies identify genomic variants associated with human traits and diseases. Most trait-associated variants are located within cell-type-specific enhancers, but the molecular mechanisms governing phenotypic variation are less well understood. Here, we show that many enhancer variants associated with red blood cell (RBC) traits map to enhancers that are co-bound by lineage-specific master transcription factors (MTFs) and signaling transcription factors (STFs) responsive to extracellular signals. The majority of enhancer variants reside on STF and not MTF motifs, perturbing DNA binding by various STFs (BMP/TGF-β-directed SMADs or WNT-induced TCFs) and affecting target gene expression. Analyses of engineered human blood cells and expression quantitative trait loci verify that disrupted STF binding leads to altered gene expression. Our results propose that the majority of the RBC-trait-associated variants that reside on transcription-factor-binding sequences fall in STF target sequences, suggesting that the phenotypic variation of RBC traits could stem from altered responsiveness to extracellular stimuli.
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http://dx.doi.org/10.1038/s41588-020-00738-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876911PMC
December 2020

Inherited causes of clonal haematopoiesis in 97,691 whole genomes.

Nature 2020 10 14;586(7831):763-768. Epub 2020 Oct 14.

Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI, USA.

Age is the dominant risk factor for most chronic human diseases, but the mechanisms through which ageing confers this risk are largely unknown. The age-related acquisition of somatic mutations that lead to clonal expansion in regenerating haematopoietic stem cell populations has recently been associated with both haematological cancer and coronary heart disease-this phenomenon is termed clonal haematopoiesis of indeterminate potential (CHIP). Simultaneous analyses of germline and somatic whole-genome sequences provide the opportunity to identify root causes of CHIP. Here we analyse high-coverage whole-genome sequences from 97,691 participants of diverse ancestries in the National Heart, Lung, and Blood Institute Trans-omics for Precision Medicine (TOPMed) programme, and identify 4,229 individuals with CHIP. We identify associations with blood cell, lipid and inflammatory traits that are specific to different CHIP driver genes. Association of a genome-wide set of germline genetic variants enabled the identification of three genetic loci associated with CHIP status, including one locus at TET2 that was specific to individuals of African ancestry. In silico-informed in vitro evaluation of the TET2 germline locus enabled the identification of a causal variant that disrupts a TET2 distal enhancer, resulting in increased self-renewal of haematopoietic stem cells. Overall, we observe that germline genetic variation shapes haematopoietic stem cell function, leading to CHIP through mechanisms that are specific to clonal haematopoiesis as well as shared mechanisms that lead to somatic mutations across tissues.
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http://dx.doi.org/10.1038/s41586-020-2819-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7944936PMC
October 2020

Inherited myeloproliferative neoplasm risk affects haematopoietic stem cells.

Nature 2020 10 14;586(7831):769-775. Epub 2020 Oct 14.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Myeloproliferative neoplasms (MPNs) are blood cancers that are characterized by the excessive production of mature myeloid cells and arise from the acquisition of somatic driver mutations in haematopoietic stem cells (HSCs). Epidemiological studies indicate a substantial heritable component of MPNs that is among the highest known for cancers. However, only a limited number of genetic risk loci have been identified, and the underlying biological mechanisms that lead to the acquisition of MPNs remain unclear. Here, by conducting a large-scale genome-wide association study (3,797 cases and 1,152,977 controls), we identify 17 MPN risk loci (P < 5.0 × 10), 7 of which have not been previously reported. We find that there is a shared genetic architecture between MPN risk and several haematopoietic traits from distinct lineages; that there is an enrichment for MPN risk variants within accessible chromatin of HSCs; and that increased MPN risk is associated with longer telomere length in leukocytes and other clonal haematopoietic states-collectively suggesting that MPN risk is associated with the function and self-renewal of HSCs. We use gene mapping to identify modulators of HSC biology linked to MPN risk, and show through targeted variant-to-function assays that CHEK2 and GFI1B have roles in altering the function of HSCs to confer disease risk. Overall, our results reveal a previously unappreciated mechanism for inherited MPN risk through the modulation of HSC function.
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http://dx.doi.org/10.1038/s41586-020-2786-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606745PMC
October 2020

In The Blood: Connecting Variant to Function In Human Hematopoiesis.

Trends Genet 2020 08 10;36(8):563-576. Epub 2020 Jun 10.

Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; Broad Institute of Harvard and Massachusetts Institute of Technology (MIT), Cambridge, MA, USA; Harvard Stem Cell Institute, Cambridge, MA, USA. Electronic address:

Genome-wide association studies (GWAS) have identified thousands of genetic variants associated with a range of human diseases and traits. However, understanding the mechanisms by which these genetic variants have an impact on associated diseases and traits, often referred to as the variant-to-function (V2F) problem, remains a significant hurdle. Solving the V2F challenge requires us to identify causative genetic variants, relevant cell types/states, target genes, and mechanisms by which variants can cause diseases or alter phenotypic traits. We discuss emerging functional approaches that are being applied to tackle the V2F problem for blood cell traits, illuminating how human genetic variation can impact on key mechanisms in hematopoiesis, as well as highlighting future prospects for this nascent field.
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http://dx.doi.org/10.1016/j.tig.2020.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363574PMC
August 2020

Infantile Myelofibrosis and Myeloproliferation with CDC42 Dysfunction.

J Clin Immunol 2020 05 17;40(4):554-566. Epub 2020 Apr 17.

Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

Studies of genetic blood disorders have advanced our understanding of the intrinsic regulation of hematopoiesis. However, such genetic studies have only yielded limited insights into how interactions between hematopoietic cells and their microenvironment are regulated. Here, we describe two affected siblings with infantile myelofibrosis and myeloproliferation that share a common de novo mutation in the Rho GTPase CDC42 (Chr1:22417990:C>T, p.R186C) due to paternal germline mosaicism. Functional studies using human cells and flies demonstrate that this CDC42 mutant has altered activity and thereby disrupts interactions between hematopoietic progenitors and key tissue microenvironmental factors. These findings suggest that further investigation of this and other related disorders may provide insights into how hematopoietic cell-microenvironment interactions play a role in human health and can be disrupted in disease. In addition, we suggest that deregulation of CDC42 may underlie more common blood disorders, such as primary myelofibrosis.
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http://dx.doi.org/10.1007/s10875-020-00778-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253386PMC
May 2020

Transcriptional States and Chromatin Accessibility Underlying Human Erythropoiesis.

Cell Rep 2019 06;27(11):3228-3240.e7

Division of Hematology/Oncology, Boston Children's Hospital, and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

Human erythropoiesis serves as a paradigm of physiologic cellular differentiation. This process is also of considerable interest for better understanding anemias and identifying new therapies. Here, we apply deep transcriptomic and accessible chromatin profiling to characterize a faithful ex vivo human erythroid differentiation system from hematopoietic stem and progenitor cells. We reveal stage-specific transcriptional states and chromatin accessibility during various stages of erythropoiesis, including 14,260 differentially expressed genes and 63,659 variably accessible chromatin peaks. Our analysis suggests differentiation stage-predominant roles for specific master regulators, including GATA1 and KLF1. We integrate chromatin profiles with common and rare genetic variants associated with erythroid cell traits and diseases, finding that variants regulating different erythroid phenotypes likely act at variable points during differentiation. In addition, we identify a regulator of terminal erythropoiesis, TMCC2, more broadly illustrating the value of this comprehensive analysis to improve our understanding of erythropoiesis in health and disease.
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http://dx.doi.org/10.1016/j.celrep.2019.05.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6579117PMC
June 2019

Gene-centric functional dissection of human genetic variation uncovers regulators of hematopoiesis.

Elife 2019 05 9;8. Epub 2019 May 9.

Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, United States.

Genome-wide association studies (GWAS) have identified thousands of variants associated with human diseases and traits. However, the majority of GWAS-implicated variants are in non-coding regions of the genome and require in depth follow-up to identify target genes and decipher biological mechanisms. Here, rather than focusing on causal variants, we have undertaken a pooled loss-of-function screen in primary hematopoietic cells to interrogate 389 candidate genes contained in 75 loci associated with red blood cell traits. Using this approach, we identify 77 genes at 38 GWAS loci, with most loci harboring 1-2 candidate genes. Importantly, the hit set was strongly enriched for genes validated through orthogonal genetic approaches. Genes identified by this approach are enriched in specific and relevant biological pathways, allowing regulators of human erythropoiesis and modifiers of blood diseases to be defined. More generally, this functional screen provides a paradigm for gene-centric follow up of GWAS for a variety of human diseases and traits.
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http://dx.doi.org/10.7554/eLife.44080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534380PMC
May 2019

Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms.

Proc Natl Acad Sci U S A 2017 01 28;114(3):E327-E336. Epub 2016 Dec 28.

Broad Institute of MIT and Harvard, Cambridge, MA 02142;

Genetic variants affecting hematopoiesis can influence commonly measured blood cell traits. To identify factors that affect hematopoiesis, we performed association studies for blood cell traits in the population-based Estonian Biobank using high-coverage whole-genome sequencing (WGS) in 2,284 samples and SNP genotyping in an additional 14,904 samples. Using up to 7,134 samples with available phenotype data, our analyses identified 17 associations across 14 blood cell traits. Integration of WGS-based fine-mapping and complementary epigenomic datasets provided evidence for causal mechanisms at several loci, including at a previously undiscovered basophil count-associated locus near the master hematopoietic transcription factor CEBPA The fine-mapped variant at this basophil count association near CEBPA overlapped an enhancer active in common myeloid progenitors and influenced its activity. In situ perturbation of this enhancer by CRISPR/Cas9 mutagenesis in hematopoietic stem and progenitor cells demonstrated that it is necessary for and specifically regulates CEBPA expression during basophil differentiation. We additionally identified basophil count-associated variation at another more pleiotropic myeloid enhancer near GATA2, highlighting regulatory mechanisms for ordered expression of master hematopoietic regulators during lineage specification. Our study illustrates how population-based genetic studies can provide key insights into poorly understood cell differentiation processes of considerable physiologic relevance.
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http://dx.doi.org/10.1073/pnas.1619052114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5255587PMC
January 2017

Systematic Functional Dissection of Common Genetic Variation Affecting Red Blood Cell Traits.

Cell 2016 Jun;165(6):1530-1545

Division of Hematology/Oncology, The Manton Center for Orphan Disease Research, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA. Electronic address:

Genome-wide association studies (GWAS) have successfully identified thousands of associations between common genetic variants and human disease phenotypes, but the majority of these variants are non-coding, often requiring genetic fine-mapping, epigenomic profiling, and individual reporter assays to delineate potential causal variants. We employ a massively parallel reporter assay (MPRA) to simultaneously screen 2,756 variants in strong linkage disequilibrium with 75 sentinel variants associated with red blood cell traits. We show that this assay identifies elements with endogenous erythroid regulatory activity. Across 23 sentinel variants, we conservatively identified 32 MPRA functional variants (MFVs). We used targeted genome editing to demonstrate endogenous enhancer activity across 3 MFVs that predominantly affect the transcription of SMIM1, RBM38, and CD164. Functional follow-up of RBM38 delineates a key role for this gene in the alternative splicing program occurring during terminal erythropoiesis. Finally, we provide evidence for how common GWAS-nominated variants can disrupt cell-type-specific transcriptional regulatory pathways.
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http://dx.doi.org/10.1016/j.cell.2016.04.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4893171PMC
June 2016

Advances in understanding erythropoiesis: evolving perspectives.

Br J Haematol 2016 Apr 5;173(2):206-18. Epub 2016 Feb 5.

Division of Hematology/Oncology, The Manton Center for Orphan Disease Research, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

Red blood cells (RBCs) are generated from haematopoietic stem and progenitor cells (HSPCs) through the step-wise process of differentiation known as erythropoiesis. In this review, we discuss our current understanding of erythropoiesis and highlight recent advances in this field. During embryonic development, erythropoiesis occurs in three distinct waves comprising first, the yolk sac-derived primitive RBCs, followed sequentially by the erythro-myeloid progenitor (EMP) and HSPC-derived definitive RBCs. Recent work has highlighted the complexity and variability that may exist in the hierarchical arrangement of progenitors responsible for erythropoiesis. Using recently defined cell surface markers, it is now possible to enrich for erythroid progenitors and precursors to a much greater extent than has been possible before. While a great deal of knowledge has been gained on erythropoiesis from model organisms, our understanding of this process is currently being refined through human genetic studies. Genes mutated in erythroid disorders can now be identified more rapidly by the use of next-generation sequencing techniques. Genome-wide association studies on erythroid traits in healthy populations have also revealed new modulators of erythropoiesis. All of these recent developments have significant promise not only for increasing our understanding of erythropoiesis, but also for improving our ability to intervene when RBC production is perturbed in disease.
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http://dx.doi.org/10.1111/bjh.13938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833665PMC
April 2016

Low-level GATA2 overexpression promotes myeloid progenitor self-renewal and blocks lymphoid differentiation in mice.

Exp Hematol 2015 Jul 20;43(7):565-77.e1-10. Epub 2015 Apr 20.

Department of Experimental Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA.

The transcription factor GATA2 is highly expressed in hematopoietic stem cells and is downregulated during lineage maturation. Gain of function mutations, loss of function mutations, and overexpression of GATA2 have been reported in acute myeloid leukemia. In previous studies, we and others showed that GATA2 overexpression at high levels, similar to that seen in hematopoietic stem cells, blocked differentiation of hematopoietic stem cells and progenitors. To better understand the effects of GATA2, we designed a Tamoxifen-inducible GATA2-estrogen receptor (ERT) vector. In the absence of Tamoxifen, small amounts of GATA2-ERT were still able to enter the nucleus in mouse bone marrow (BM) cells, providing us with a tool to test the effects of low-level GATA2 overexpression. We observed that this low-level GATA2 overexpression enhanced self-renewal of myeloid progenitors in vitro and resulted in immortalization of BM cells to myeloid cell lines. Continuous GATA2-ERT expression was required for the proliferation of these immortalized lines. Myeloid expansion and a block in T and B lineage differentiation were observed in mice transplanted with GATA2-ERT-expressing BM cells. Myeloid expansion occurred after the granulocyte monocyte progenitor stage, and lymphoid block was distal to the common lymphoid progenitor in transgenic mice. GATA2 appeared to induce growth via downstream activation of Nmyc and Hoxa9. Our results demonstrate that GATA2 overexpression at low level confers self-renewal capacity to myeloid progenitors and is relevant to myeloid leukemia development.
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http://dx.doi.org/10.1016/j.exphem.2015.04.002DOI Listing
July 2015
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