Publications by authors named "Sjoerd Holwerda"

14 Publications

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ZNRF3 and RNF43 cooperate to safeguard metabolic liver zonation and hepatocyte proliferation.

Cell Stem Cell 2021 Jun 11. Epub 2021 Jun 11.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland. Electronic address:

AXIN2 and LGR5 mark intestinal stem cells (ISCs) that require WNT/β-Catenin signaling for constant homeostatic proliferation. In contrast, AXIN2/LGR5+ pericentral hepatocytes show low proliferation rates despite a WNT/β-Catenin activity gradient required for metabolic liver zonation. The mechanisms restricting proliferation in AXIN2+ hepatocytes and metabolic gene expression in AXIN2+ ISCs remained elusive. We now show that restricted chromatin accessibility in ISCs prevents the expression of β-Catenin-regulated metabolic enzymes, whereas fine-tuning of WNT/β-Catenin activity by ZNRF3 and RNF43 restricts proliferation in chromatin-permissive AXIN2+ hepatocytes, while preserving metabolic function. ZNRF3 deletion promotes hepatocyte proliferation, which in turn becomes limited by RNF43 upregulation. Concomitant deletion of RNF43 in ZNRF3 mutant mice results in metabolic reprogramming of periportal hepatocytes and induces clonal expansion in a subset of hepatocytes, ultimately promoting liver tumors. Together, ZNRF3 and RNF43 cooperate to safeguard liver homeostasis by spatially and temporally restricting WNT/β-Catenin activity, balancing metabolic function and hepatocyte proliferation.
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http://dx.doi.org/10.1016/j.stem.2021.05.013DOI Listing
June 2021

Postmitotic Hoxa5 Expression Specifies Pontine Neuron Positional Identity and Input Connectivity of Cortical Afferent Subsets.

Cell Rep 2020 06;31(11):107767

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland; University of Basel, 4051 Basel, Switzerland. Electronic address:

The mammalian precerebellar pontine nucleus (PN) has a main role in relaying cortical information to the cerebellum. The molecular determinants establishing ordered connectivity patterns between cortical afferents and precerebellar neurons are largely unknown. We show that expression of Hox5 transcription factors is induced in specific subsets of postmitotic PN neurons at migration onset. Hox5 induction is achieved by response to retinoic acid signaling, resulting in Jmjd3-dependent derepression of Polycomb chromatin and 3D conformational changes. Hoxa5 drives neurons to settle posteriorly in the PN, where they are monosynaptically targeted by cortical neuron subsets mainly carrying limb somatosensation. Furthermore, Hoxa5 postmigratory ectopic expression in PN neurons is sufficient to attract cortical somatosensory inputs regardless of position and avoid visual afferents. Transcriptome analysis further suggests that Hoxa5 is involved in circuit formation. Thus, Hoxa5 coordinates postmitotic specification, migration, settling position, and sub-circuit assembly of PN neuron subsets in the cortico-cerebellar pathway.
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http://dx.doi.org/10.1016/j.celrep.2020.107767DOI Listing
June 2020

AXIN2 Pericentral Hepatocytes Have Limited Contributions to Liver Homeostasis and Regeneration.

Cell Stem Cell 2020 01 19;26(1):97-107.e6. Epub 2019 Dec 19.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland. Electronic address:

The existence of specialized liver stem cell populations, including AXIN2 pericentral hepatocytes, that safeguard homeostasis and repair has been controversial. Here, using AXIN2 lineage tracing in BAC-transgenic mice, we confirm the regenerative potential of intestinal stem cells (ISCs) but find limited roles for pericentral hepatocytes in liver parenchyma homeostasis. Liver regrowth following partial hepatectomy is enabled by proliferation of hepatocytes throughout the liver, rather than by a pericentral population. Periportal hepatocyte injury triggers local repair as well as auxiliary proliferation in all liver zones. DTA-mediated ablation of AXIN2 pericentral hepatocytes transiently disrupts this zone, which is reestablished by conversion of pericentral vein-juxtaposed glutamine synthetase (GS) hepatocytes into GS hepatocytes and by compensatory proliferation of hepatocytes across liver zones. These findings show hepatocytes throughout the liver can upregulate AXIN2 and LGR5 after injury and contribute to liver regeneration on demand, without zonal dominance by a putative pericentral stem cell population.
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http://dx.doi.org/10.1016/j.stem.2019.10.011DOI Listing
January 2020

PAX8 activates metabolic genes via enhancer elements in Renal Cell Carcinoma.

Nat Commun 2019 08 20;10(1):3739. Epub 2019 Aug 20.

Disease Area Oncology, Novartis Institute for Biomedical Research, Basel, Switzerland.

Transcription factor networks shape the gene expression programs responsible for normal cell identity and pathogenic state. Using Core Regulatory Circuitry analysis (CRC), we identify PAX8 as a candidate oncogene in Renal Cell Carcinoma (RCC) cells. Validation of large-scale functional genomic screens confirms that PAX8 silencing leads to decreased proliferation of RCC cell lines. Epigenomic analyses of PAX8-dependent cistrome demonstrate that PAX8 largely occupies active enhancer elements controlling genes involved in various metabolic pathways. We selected the ferroxidase Ceruloplasmin (CP) as an exemplary gene to dissect PAX8 molecular functions. PAX8 recruits histone acetylation activity at bound enhancers looping onto the CP promoter. Importantly, CP expression correlates with sensitivity to PAX8 silencing and identifies a subset of RCC cases with poor survival. Our data identifies PAX8 as a candidate oncogene in RCC and provides a potential biomarker to monitor its activity.
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http://dx.doi.org/10.1038/s41467-019-11672-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702156PMC
August 2019

The formation of the light-sensing compartment of cone photoreceptors coincides with a transcriptional switch.

Elife 2017 11 6;6. Epub 2017 Nov 6.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

High-resolution daylight vision is mediated by cone photoreceptors. The molecular program responsible for the formation of their light sensor, the outer segment, is not well understood. We correlated daily changes in ultrastructure and gene expression in postmitotic mouse cones, between birth and eye opening, using serial block-face electron microscopy (EM) and RNA sequencing. Outer segments appeared rapidly at postnatal day six and their appearance coincided with a switch in gene expression. The switch affected over 14% of all expressed genes. Genes that switched off were rich in transcription factors and neurogenic genes. Those that switched on contained genes relevant for cone function. Chromatin rearrangements in enhancer regions occurred before the switch was completed, but not after. We provide a resource comprised of correlated EM, RNAseq, and ATACseq data, showing that the growth of a key compartment of a postmitotic cell involves an extensive switch in gene expression and chromatin accessibility.
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http://dx.doi.org/10.7554/eLife.31437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5685475PMC
November 2017

Gene bivalency at Polycomb domains regulates cranial neural crest positional identity.

Science 2017 03;355(6332)

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4051 Basel, Switzerland.

The cranial neural crest cells are multipotent cells that provide head skeletogenic mesenchyme and are crucial for craniofacial patterning. We analyzed the chromatin landscapes of mouse cranial neural crest subpopulations in vivo. Early postmigratory subpopulations contributing to distinct mouse craniofacial structures displayed similar chromatin accessibility patterns yet differed transcriptionally. Accessible promoters and enhancers of differentially silenced genes carried H3K27me3/H3K4me2 bivalent chromatin marks embedded in large -dependent Polycomb domains, indicating transcriptional poising. These postmigratory bivalent chromatin regions were already present in premigratory progenitors. At Polycomb domains, H3K27me3 antagonized H3K4me2 deposition, which was restricted to accessible sites. Thus, bivalent Polycomb domains provide a chromatin template for the regulation of cranial neural crest cell positional identity in vivo, contributing insights into the epigenetic regulation of face morphogenesis.
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http://dx.doi.org/10.1126/science.aal2913DOI Listing
March 2017

Small chromosomal regions position themselves autonomously according to their chromatin class.

Genome Res 2017 06 24;27(6):922-933. Epub 2017 Mar 24.

Department of Biology II, Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany.

The spatial arrangement of chromatin is linked to the regulation of nuclear processes. One striking aspect of nuclear organization is the spatial segregation of heterochromatic and euchromatic domains. The mechanisms of this chromatin segregation are still poorly understood. In this work, we investigated the link between the primary genomic sequence and chromatin domains. We analyzed the spatial intranuclear arrangement of a human artificial chromosome (HAC) in a xenospecific mouse background in comparison to an orthologous region of native mouse chromosome. The two orthologous regions include segments that can be assigned to three major chromatin classes according to their gene abundance and repeat repertoire: (1) gene-rich and SINE-rich euchromatin; (2) gene-poor and LINE/LTR-rich heterochromatin; and (3) gene-depleted and satellite DNA-containing constitutive heterochromatin. We show, using fluorescence in situ hybridization (FISH) and 4C-seq technologies, that chromatin segments ranging from 0.6 to 3 Mb cluster with segments of the same chromatin class. As a consequence, the chromatin segments acquire corresponding positions in the nucleus irrespective of their chromosomal context, thereby strongly suggesting that this is their autonomous property. Interactions with the nuclear lamina, although largely retained in the HAC, reveal less autonomy. Taken together, our results suggest that building of a functional nucleus is largely a self-organizing process based on mutual recognition of chromosome segments belonging to the major chromatin classes.
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http://dx.doi.org/10.1101/gr.213751.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453326PMC
June 2017

CTCF Binding Polarity Determines Chromatin Looping.

Mol Cell 2015 Nov 29;60(4):676-84. Epub 2015 Oct 29.

Hubrecht Institute-KNAW and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands. Electronic address:

CCCTC-binding factor (CTCF) is an architectural protein involved in the three-dimensional (3D) organization of chromatin. In this study, we assayed the 3D genomic contact profiles of a large number of CTCF binding sites with high-resolution 4C-seq. As recently reported, our data also suggest that chromatin loops preferentially form between CTCF binding sites oriented in a convergent manner. To directly test this, we used CRISPR/Cas9 genome editing to delete core CTCF binding sites in three loci, including the CTCF site in the Sox2 super-enhancer. In all instances, CTCF and cohesin recruitment were lost, and chromatin loops with distal, convergent CTCF sites were disrupted or destabilized. Re-insertion of oppositely oriented CTCF recognition sequences restored CTCF and cohesin recruitment, but did not re-establish chromatin loops. We conclude that CTCF binding polarity plays a functional role in the formation of higher-order chromatin structure.
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http://dx.doi.org/10.1016/j.molcel.2015.09.023DOI Listing
November 2015

Flexible long-range loops in the VH gene region of the Igh locus facilitate the generation of a diverse antibody repertoire.

Immunity 2013 Aug;39(2):229-44

Research Institute of Molecular Pathology, Vienna Biocenter, Dr. Bohr-Gasse 7, 1030 Vienna, Austria.

The immunoglobulin heavy-chain (Igh) locus undergoes large-scale contraction in pro-B cells, which facilitates VH-DJH recombination by juxtaposing distal VH genes next to the DJH-rearranged gene segment in the 3' proximal Igh domain. By using high-resolution mapping of long-range interactions, we demonstrate that local interaction domains established the three-dimensional structure of the extended Igh locus in lymphoid progenitors. In pro-B cells, these local domains engaged in long-range interactions across the Igh locus, which depend on the regulators Pax5, YY1, and CTCF. The large VH gene cluster underwent flexible long-range interactions with the more rigidly structured proximal domain, which probably ensures similar participation of all VH genes in VH-DJH recombination to generate a diverse antibody repertoire. These long-range interactions appear to be an intrinsic feature of the VH gene cluster, because they are still generated upon mutation of the Eμ enhancer, IGCR1 insulator, or 3' regulatory region in the proximal Igh domain.
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http://dx.doi.org/10.1016/j.immuni.2013.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810778PMC
August 2013

Allelic exclusion of the immunoglobulin heavy chain locus is independent of its nuclear localization in mature B cells.

Nucleic Acids Res 2013 Aug 7;41(14):6905-16. Epub 2013 Jun 7.

Hubrecht Institute-KNAW & University Medical Center Utrecht, Utrecht 3584 CT, The Netherlands.

In developing B cells, the immunoglobulin heavy chain (IgH) locus is thought to move from repressive to permissive chromatin compartments to facilitate its scheduled rearrangement. In mature B cells, maintenance of allelic exclusion has been proposed to involve recruitment of the non-productive IgH allele to pericentromeric heterochromatin. Here, we used an allele-specific chromosome conformation capture combined with sequencing (4C-seq) approach to unambigously follow the individual IgH alleles in mature B lymphocytes. Despite their physical and functional difference, productive and non-productive IgH alleles in B cells and unrearranged IgH alleles in T cells share many chromosomal contacts and largely reside in active chromatin. In brain, however, the locus resides in a different repressive environment. We conclude that IgH adopts a lymphoid-specific nuclear location that is, however, unrelated to maintenance of allelic exclusion. We additionally find that in mature B cells-but not in T cells-the distal VH regions of both IgH alleles position themselves away from active chromatin. This, we speculate, may help to restrict enhancer activity to the productively rearranged VH promoter element.
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http://dx.doi.org/10.1093/nar/gkt491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3737562PMC
August 2013

CTCF: the protein, the binding partners, the binding sites and their chromatin loops.

Philos Trans R Soc Lond B Biol Sci 2013 6;368(1620):20120369. Epub 2013 May 6.

Hubrecht Institute-KNAW and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.

CTCF has it all. The transcription factor binds to tens of thousands of genomic sites, some tissue-specific, others ultra-conserved. It can act as a transcriptional activator, repressor and insulator, and it can pause transcription. CTCF binds at chromatin domain boundaries, at enhancers and gene promoters, and inside gene bodies. It can attract many other transcription factors to chromatin, including tissue-specific transcriptional activators, repressors, cohesin and RNA polymerase II, and it forms chromatin loops. Yet, or perhaps therefore, CTCF's exact function at a given genomic site is unpredictable. It appears to be determined by the associated transcription factors, by the location of the binding site relative to the transcriptional start site of a gene, and by the site's engagement in chromatin loops with other CTCF-binding sites, enhancers or gene promoters. Here, we will discuss genome-wide features of CTCF binding events, as well as locus-specific functions of this remarkable transcription factor.
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http://dx.doi.org/10.1098/rstb.2012.0369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682731PMC
December 2013

Chromatin loops, gene positioning, and gene expression.

Front Genet 2012 17;3:217. Epub 2012 Oct 17.

Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences, University Medical Center Utrecht Utrecht, Netherlands.

Technological developments and intense research over the last years have led to a better understanding of the 3D structure of the genome and its influence on genome function inside the cell nucleus. We will summarize topological studies performed on four model gene loci: the α- and β-globin gene loci, the antigen receptor loci, the imprinted H19-Igf2 locus and the Hox gene clusters. Collectively, these studies show that regulatory DNA sequences physically contact genes to control their transcription. Proteins set up the 3D configuration of the genome and we will discuss the roles of the key structural organizers CTCF and cohesin, the nuclear lamina and the transcription machinery. Finally, genes adopt non-random positions in the nuclear interior. We will review studies on gene positioning and propose that cell-specific genome conformations can juxtapose a regulatory sequence on one chromosome to a responsive gene on another chromosome to cause altered gene expression in subpopulations of cells.
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http://dx.doi.org/10.3389/fgene.2012.00217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3473233PMC
October 2012

Robust 4C-seq data analysis to screen for regulatory DNA interactions.

Nat Methods 2012 Oct 9;9(10):969-72. Epub 2012 Sep 9.

Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Utrecht, The Netherlands.

Regulatory DNA elements can control the expression of distant genes via physical interactions. Here we present a cost-effective methodology and computational analysis pipeline for robust characterization of the physical organization around selected promoters and other functional elements using chromosome conformation capture combined with high-throughput sequencing (4C-seq). Our approach can be multiplexed and routinely integrated with other functional genomics assays to facilitate physical characterization of gene regulation.
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http://dx.doi.org/10.1038/nmeth.2173DOI Listing
October 2012

4C technology: protocols and data analysis.

Methods Enzymol 2012 ;513:89-112

Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands.

Chromosome conformation capture (3C) technology and its genome-wide derivatives have revolutionized our knowledge on chromatin folding and nuclear organization. 4C-seq Technology combines 3C principles with high-throughput sequencing (4C-seq) to enable for unbiased genome-wide screens for DNA contacts made by single genomic sites of interest. Here, we discuss in detail the design, application, and data analysis of 4C-seq experiments. Based on many hundreds of different 4C-seq experiments, we define criteria to assess data quality and show how different restriction enzymes and cross-linking conditions affect results. We describe in detail the mapping strategy of 4C-seq reads and show advanced strategies for data analysis.
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http://dx.doi.org/10.1016/B978-0-12-391938-0.00004-5DOI Listing
December 2012
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