scholarly journals Isolated Changes in Chromatin Accessibility and Enhancer-Promoter Contacts at the β-Globin Locus Distinguish Fetal Hemoglobin Producing F-Cells from a-Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 855-855
Author(s):  
Eugene Khandros ◽  
Peng Huang ◽  
Scott A. Peslak ◽  
Malini Sharma ◽  
Osheiza Abdulmalik ◽  
...  
Keyword(s):  
Long Range ◽  
Healthy Donor ◽  
Chromatin Accessibility ◽  
Global Changes ◽  
Epigenetic Variation ◽  
Gene Promoters ◽  
Globin Genes ◽  
Chromatin Architecture ◽  
A Cells ◽  
F Cells

Abstract Reversal of the developmental switch from fetal (HbF, α 2γ 2) to adult (HbA,α 2β 2) hemoglobin is an important therapeutic approach for sickle cell disease (SCD) and β-thalassemia. It has been noted since the 1950s that a small number of circulating red blood cells, called F-cells, produce elevated levels of HbF; these cells are resistant to sickling and are present in increased numbers in patients with SCD and those treated with pharmacological HbF inducers such as hydroxyurea. Because successful therapy for SCD requires increasing the number of F-cells, it is imperative to understand how these cells arise. This can potentially occur through a shift towards a global fetal-like program, selective variation in levels of known HbF silencers such as BCL11A or LRF, or through discrete epigenetic changes at the β-globin locus. We previously began to address this clinically important question using a novel experimental approach of sorting cultured primary human erythroblasts into HbF-high (F-cell) and HbF-low (A-cell) populations (Khandros et al, Blood 2020). We showed that surprisingly, F-cells from healthy donor primary erythroid cultures have minimal transcriptional differences with A-cells. Unexpectedly, this was also the case when comparing responders (F-cells) and non-responders (A-cells) to treatment with the HbF inducers pomalidomide and hydroxyurea, and there were no differences in the expression of known HbF regulators. We therefore hypothesize that HbF synthesis in F-cells is determined by epigenetic variation confined to the β-globin locus (and not by global changes in the cell fate or nuclear milieu). To test this hypothesis, we compared genome wide chromatin accessibility by Assay for Transposase-Accessible Chromatin (ATAC-seq) in differentiation stage-matched F- and A-cells from healthy donor primary erythroid cultures, treated with vehicle, hydroxyurea, or pomalidomide. We observed striking similarities between F- and A-cells: out of 83,295 peaks called across all conditions, a mere five regions of differential accessibility were found, all at the β-globin locus (at the promoters and 3' UTR regions of the HBG1 and HBG2 genes as well as the BGLT3 non-coding RNA and HBBP1 pseudogene). This remarkable similarity in the global chromatin landscape between A- and F-cells cements the notion that these cells are fundamentally the same in terms of developmental and differentiation states, and that local epigenetic variation at the β-globin locus underlies the differences in HbF production. We also found that the gains in ATAC signal at the HBG1/2 genes were the most pronounced in F-cells from pomalidomide treated cultures, consistent with our finding that F-cells that arise following pomalidomide treatment have a higher content of HBG1/2 transcripts per cell. Drug treatments led to a larger number of changes in ATAC-seq peaks, at 123 and 1015 sites for treatment with hydroxyurea or pomalidomide, respectively, compared to vehicle. However, since differences at only 5 ATAC-seq peaks were observed between between F- and A-cells, we infer that the broader changes upon drug treatment are not needed for the phenotypic differences between F- and A-cells. Since transcription of the β-type globin genes is controlled by developmental stage-specific long-range contacts between the gene promoters and the locus control region (LCR), we determined whether the increase chromatin accessibility at the γ-globin genes in F-cells was associated with enhanced contacts with the LCR. Capture-C experiments revealed increased LCR-HBG1/2 promoter contacts and reduced LCR contacts with the adult HBB and HBD promoters in F-cells vs A-cells, demonstrating that local gains in chromatin accessibility are linked to long-range enhancer promoter contacts. Additionally, we did not detect differences in long-range chromatin contacts at several developmentally regulated genes, including LIN28B and BCL11A, solidifying the idea that γ-globin production in F-cells is specified locally through chromatin accessibility and chromatin architecture. In sum, our studies demonstrate that in adults, F-cells do not arise through reversion to a fetal like state or variation in expression of any known HbF regulator. Rather these cells reflect highly localized, perhaps stochastic modulation of chromatin architecture at the β-globin locus. Disclosures Blobel: Fulcrum Therapeutics, Inc.: Consultancy; Pfizer: Consultancy.

scholarly journals A β-Globin Locus-Intrinsic Epigenetic Mechanism Underlies Fetal Globin Production in F-Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 16-17
Author(s):  
Eugene Khandros ◽  
Peng Huang ◽  
Scott A. Peslak ◽  
Malini Sharma ◽  
Osheiza Abdulmalik ◽  
...  
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Epigenetic Mechanism ◽  
Chromatin Domain ◽  
Globin Genes ◽  
Chromatin Architecture ◽  
A Cells ◽  
F Cells ◽  
Globin Gene Expression

Upregulation of fetal hemoglobin (HbF, α2γ2) by reversing the developmental switch to adult HbA (α2β2) is a key approach for both pharmacologic and gene targeting therapies in the treatment of sickle cell disease (SCD) and β-thalassemia. HbF expression in healthy individuals, patients with SCD, and those treated with hydroxyurea is restricted to a subset of red blood cells known as F-cells; effective SCD therapy requires increasing the proportion of F-cells expressing sufficient HbF to block sickling. Although these cells have been observed since the 1950s, there have not been previous direct comparisons of F-cells to matched HbF-low A-cells from the same individual. Fetal erythroblasts have distinct global transcriptional programs and distinct long-range chromatin looping at the β-globin locus when compared to adult erythroblasts. An important question is therefore whether F-cells are formed through reversion to a fetal-like state at the transcriptional and epigenetic level. To address this clinically important question, we previously reported development of new techniques for the purification of stage-matched F- and A-erythroblasts from primary human CD34+ cell erythroid cultures and their downstream analysis (Khandros et al, Blood 2020). We demonstrated that F-cells in primary erythroid cultures have minimal transcriptional differences with A-cells and that the few differentially expressed transcripts do not overlap with fetal-specific transcripts. Furthermore, treatment with hydroxyurea or pomalidomide did not enhance transcriptional differences between F- and A-cells. Surprisingly, we did not find differences in the expression of any known HbF regulators such as BCL11A, LRF, or NuRD complex members that would account for differential HbF expression. Based on these findings, we hypothesized that F-cells are distinguished by epigenetic variation specifically at the β-globin locus. Given that fetal erythroblasts differ from adult erythroblasts in the chromatin architecture of the β-globin locus (e.g. Huang et al, Genes and Development 2017), we compared the higher order chromatin organization of the β-globin locus between F- and A-cells by Capture-C, a next-generation sequencing-adapted form of chromatin conformation capture. We found that in F-cells, contacts between the distal enhancer and the promoters of the fetal globin genes HBG1 and HBG2 were increased, while those between the enhancer and adult globin genes (HBB and HBD) were reduced. Other architectural changes associated with fetal globin gene expression, including fetal specific contacts of an intergenic non-coding gene with chromatin domain boundaries at the β-globin locus were also partially enriched in F-cells. We also did not find any differences in promoter-enhancer contacts between F- and A-cells for other developmentally regulated genes BCL11A, LIN28B, and THRB. Together these results are consistent with the concept that epigenetic changes associated with nuclear architecture that occur specifically at the β-globin locus underlie the difference in globin gene expression profiles between F- and A-cells. In sum our data demonstrate that in adult erythropoiesis, F-cells do not arise through either a wholesale reversion to a fetal-like genetic program or through variation in any known HbF regulators. Instead, modulation of chromatin architecture intrinsic to the β-globin locus, perhaps in a stochastic manner, accounts for elevated fetal globin expression in F-cells. We are currently performing mechanistic studies to elucidate the basis for the epigenetic regulation of the β-globin locus in F-cells. These studies will further our understanding of fetal hemoglobin regulation in adult cells and might inform new therapeutic approaches for SCD and β-thalassemia. Disclosures Blobel: Fulcrum Therapeutics: Consultancy; Pfizer: Research Funding.

scholarly journals Understanding heterogeneity of fetal hemoglobin induction through comparative analysis of F and A erythroblasts

Blood ◽  
2020 ◽  
Vol 135 (22) ◽  
pp. 1957-1968 ◽  
Author(s):  
Eugene Khandros ◽  
Peng Huang ◽  
Scott A. Peslak ◽  
Malini Sharma ◽  
Osheiza Abdulmalik ◽  
...  
Keyword(s):  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Transcriptional Level ◽  
Globin Genes ◽  
Proteomic Profiling ◽  
A Cells ◽  
F Cells ◽  
Adult Hemoglobin ◽  
Differentiation Stage ◽  
Developmental Switch

Abstract Reversing the developmental switch from fetal hemoglobin (HbF, α2γ2) to adult hemoglobin (HbA, α2β2) is an important therapeutic approach in sickle cell disease (SCD) and β-thalassemia. In healthy individuals, SCD patients, and patients treated with pharmacologic HbF inducers, HbF is present only in a subset of red blood cells known as F cells. Despite more than 50 years of observations, the cause for this heterocellular HbF expression pattern, even among genetically identical cells, remains unknown. Adult F cells might represent a reversion of a given cell to a fetal-like epigenetic and transcriptional state. Alternatively, isolated transcriptional or posttranscriptional events at the γ-globin genes might underlie heterocellularity. Here, we set out to understand the heterogeneity of HbF activation by developing techniques to purify and profile differentiation stage-matched late erythroblast F cells and non–F cells (A cells) from the human HUDEP2 erythroid cell line and primary human erythroid cultures. Transcriptional and proteomic profiling of these cells demonstrated very few differences between F and A cells at the RNA level either under baseline conditions or after treatment with HbF inducers hydroxyurea or pomalidomide. Surprisingly, we did not find differences in expression of any known HbF regulators, including BCL11A or LRF, that would account for HbF activation. Our analysis shows that F erythroblasts are not significantly different from non-HbF–expressing cells and that the primary differences likely occur at the transcriptional level at the β-globin locus.

scholarly journals Understanding Heterogeneity of Fetal Hemoglobin Induction through Comparative Analysis of Stage-Matched F- and a-Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 981-981
Author(s):  
Eugene Khandros ◽  
Peng Huang ◽  
Scott A. Peslak ◽  
Belinda Giardine ◽  
Zhe Zhang ◽  
...  
Keyword(s):  
Research Funding ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Differentially Expressed ◽  
Erythroid Cell ◽  
Alternative Mechanism ◽  
Globin Genes ◽  
A Cells ◽  
Hydroxyurea Treatment ◽  
F Cells

Reversing the developmental switch from fetal (HbF, α2γ2) to adult (HbA, α2β2) hemoglobin is an important therapeutic approach in sickle cell disease (SCD) and β-thalassemia. Elevated HbF levels due to genetic variation or through therapeutic induction by hydroxyurea (HU) attenuate the severity of both disorders. HbF in healthy individuals, SCD patients, and patients treated with HU is present in a heterocellular fashion in a subset of red blood cells known as F-cells. Despite over 50 years of observations of F-cells, it is not known why only some cells in a genetically identical population are able to express HbF or respond to pharmacological inducers. Adult F-cells can potentially represent a reversion to a fetal-like epigenetic and transcriptional program, or alternatively isolated transcriptional or posttranscriptional events at the γ-globin genes. Here we set out to understand the heterogeneity of HbF activation and gain insights into whether the mechanisms underlying the heterocellular response are similar or distinct in response to different HbF inducers. To this end we developed techniques to purify differentiation stage-matched late erythroblast F-cells and non-F cells (A-cells) from the human HUDEP2 erythroid cell line and primary CD34 cell erythroid cultures using a reversible fixation protocol enabling extraction of high-quality RNA and protein. Purified F-cells from both sources were enriched for γ-globin transcripts by 200-500 fold by RT-PCR, validating the purification scheme. We profiled these cells by RNA-seq using a modified method that depletes globin mRNAs and ribosomal RNAs and is capable of detecting low abundance transcripts, as well as by mass spectrometry using size fractionation to increase the number of detected proteins. In differentiated clonal HUDEP2 cells, differences between F-cells and A-cells were remarkably small, with only 62 differentially expressed transcripts and 20 differentially expressed proteins. Top differentially expressed transcripts were γ-globin and the non-coding β-globin locus transcripts BGLT3 and HBBP1. Interestingly, there were no significant changes in known HbF regulators BCL11A, LRF, and HRI at the RNA or protein level. Gene set enrichment analysis (GSEA) using a previously generated set of differentially expressed transcripts from adult and fetal-derived CD34 erythroid cultures showed enrichment of fetal transcripts in F-cells and adult transcripts in A-cells. We also carried out transcriptome analysis of sorted matched late erythroblast F-cells and A-cells from human CD34+ cell erythroid cultures at different time points. Similar to HUDEP2 cells, only small numbers of transcripts were differentially expressed (33 at 8 days, 17 at 11 days, and 261 at 14 days). BCL11A, LRF, and HRI were not differentially expressed at the earlier timepoints, and BCL11A and HRI were at most decreased by about 20% at the 14-day mark. GSEA analysis did not show fetal transcript enrichment in day 8. At days 11 and 14, there was some enrichment of fetal transcripts in F-cells but not to the degree of HUDEP2 cells. Finally, we analyzed sorted F- and A-cells from day 11 CD34+ erythroid cultures treated with hydroxyurea and pomalidomide. Again, differences between F- and A-cells were small with hydroxyurea treatment (53 transcripts) and more significant with pomalidomide treatment (400 transcripts). We have successfully established an approach to analyze stage-matched γ-globin containing cells from a genetically identical starting population, with high degree of enrichment. Our preliminary data indicate that these cells are overall highly similar to non-γ-containing cells, but do show some enrichment of fetal-specific transcripts, more so in HUDEP2 cells. The differences between F- and A- cells are overall smaller than those observed by us and others in profiling of fetal and adult-derived erythroblasts. This suggests that F-cells are not formed by reversion to a fetal-like state but rather through specific changes at the β-globin locus. Importantly, we do not find differential levels of any known γ-globin regulators, suggesting an alternative mechanism for the heterocellular expression pattern. Studies are currently ongoing to carry out epigenetic profiling of F-cells. Disclosures Blobel: Bioverativ: Research Funding; Pfizer: Research Funding.

Mechanistic Insights into Forced Chromatin Looping Mediated Activation of Fetal Globin Gene Expression

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 331-331
Author(s):  
Caroline Bartman ◽  
Gerd A. Blobel ◽  
Jeremy Grevet ◽  
Chris C.S. Hsiung ◽  
Jeremy W. Rupon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Gene Promoters ◽  
Globin Genes ◽  
Chromatin Architecture ◽  
Chromatin Looping ◽  
Globin Promoter

Abstract The β-globin enhancer, called locus control region (LCR), is required for high level expression of all b-type globin genes. The LCR is in physical proximity with the genes it controls, with contacts shifting from embryonic (ε) to fetal (γ) and finally to adult (δ and β) globin gene promoters during development. In prior studies we showed that forced chromatin contacts between the LCR and the β-globin promoter led to transcriptional activation, suggesting that LCR-promoter looping causally underlies β-globin transcription (Deng et al. Cell 2012). In these studies, the transcription co-factor Ldb1 was tethered to the β-globin promoter using artificial zinc finger (ZF) DNA binding proteins, to trigger the promoter-LCR interaction. We subsequently showed that tethering Ldb1 to the promoters of developmentally silenced embryonic or fetal globin genes reactivated their expression in adult erythroblasts in a manner dependent on looped contacts with the LCR (Deng et al. Cell 2014). This work established a novel strategy to raise fetal globin expression in patients with sickle cell anemia. To examine mechanistically the effects of chromatin looping on gene expression we performed single molecule RNA FISH experiments to precisely measure transcription output at individual alleles before and after enforced LCR-γ-globin looping. The experiments were carried out in primary human erythroblasts, which produce elevated levels of γ-globin when exposed to culture conditions. Preliminary data suggest that the majority of transcripts emerge from the β-globin gene with a smaller fraction of transcripts coming from the γ-globin gene as expected. Among cells producing any type of globin primary transcripts, a significant fraction (25%-35%) of cells co-express γ- and β-globin. Importantly, γ- and β-globin are frequently transcribed from the same allele. Forced juxtaposition of the LCR and the γ-globin promoter increases the number of alleles expressing only γ-globin while reducing the number of alleles expressing only the β-globin gene. This result is consistent with the γ- and β-globin genes competing for LCR activity, and emphasizes the usefulness of this approach in the context of sickle cell anemia in which not only elevated levels of fetal hemoglobin but also reduction of the mutant hemoglobin are desirable. Surprisingly, however, the proportion of alleles co-expressing γ-globin and β-globin remains largely constant. We are testing whether co-expression from the same allele is LCR independent. Finally, our studies suggest that LCR-promoter contacts increase the probability of transcription of a given allele. We will also present work addressing the critical question as to how alteration of chromatin architecture overcomes the action of transcriptional repressive complexes, such as Bcl11a, which normally maintain embryonic and fetal globin genes in a repressed state throughout adulthood. In sum, our studies produce a deeper understanding of the interplay of chromatin architecture and gene expression in a system that holds great potential for therapeutic application in patients with hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Leveraging the Mendelian Disorders of the Epigenetic Machinery to Systematically Map Functional Epigenetic Variation

2020 ◽  
Author(s):  
Teresa R. Luperchio ◽  
Leandros Boukas ◽  
Li Zhang ◽  
Genay O. Pilarowski ◽  
Jenny Jiang ◽  
...  
Keyword(s):  
Multiple Testing ◽  
Iga Deficiency ◽  
Chromatin Accessibility ◽  
Epigenetic Variation ◽  
Gene Promoters ◽  
Loss Of Function ◽  
Causative Gene ◽  
Mendelian Disorders ◽  
Epigenetic Machinery

AbstractThe Mendelian Disorders of the Epigenetic Machinery (MDEMs) have emerged as a class of Mendelian disorders caused by loss-of-function variants in epigenetic regulators. Although each MDEM has a different causative gene, they exhibit several overlapping disease manifestations. Here, we hypothesize that this phenotypic convergence is a consequence of common abnormalities at the epigenomic level, which directly or indirectly lead to downstream convergence at the transcriptomic level. Therefore, we seek to identify abnormalities shared across multiple MDEMs, in order to pinpoint locations where epigenetic variation is causally related to disease phenotypes. To this end, we perform a comprehensive interrogation of chromatin (ATAC-Seq) and expression (RNA-Seq) states in B cells from mouse models of three MDEMs (Kabuki types 1&2 and Rubinstein-Taybi syndromes). We build on recent work in covariate-powered multiple testing to develop a new approach for the overlap analysis, which enables us to find extensive overlap primarily localized in gene promoters. We show that disruption of chromatin accessibility at promoters often leads to disruption of downstream gene expression, and identify 463 loci and 249 genes with shared disruption across all three MDEMs. As an example of how widespread dysregulation leads to specific phenotypes, we show that subtle expression alterations of multiple, IgA-relevant genes, collectively contribute to IgA deficiency in KS1 and RT1. In contrast, we predict that KS2 does not have IgA deficiency, and confirm this observation in vivo. We propose that the joint study of MDEMs offers a principled approach for systematically mapping functional epigenetic variation in mammals.

scholarly journals Leveraging the Mendelian disorders of the epigenetic machinery to systematically map functional epigenetic variation

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Teresa Romeo Luperchio ◽  
Leandros Boukas ◽  
Li Zhang ◽  
Genay Pilarowski ◽  
Jenny Jiang ◽  
...  
Keyword(s):  
Iga Deficiency ◽  
Chromatin Accessibility ◽  
Epigenetic Variation ◽  
Gene Promoters ◽  
Epigenetic Alterations ◽  
Rna Seq ◽  
Causative Gene ◽  
Epigenetic Machinery ◽  
Overlap Analysis

Although each Mendelian Disorder of the Epigenetic Machinery (MDEM) has a different causative gene, there are shared disease manifestations. We hypothesize that this phenotypic convergence is a consequence of shared epigenetic alterations. To identify such shared alterations, we interrogate chromatin (ATAC-seq) and expression (RNA-seq) states in B cells from three MDEM mouse models (Kabuki [KS] type 1 and 2 and Rubinstein-Taybi type 1 [RT1] syndromes). We develop a new approach for the overlap analysis and find extensive overlap primarily localized in gene promoters. We show that disruption of chromatin accessibility at promoters often disrupts downstream gene expression, and identify 587 loci and 264 genes with shared disruption across all three MDEMs. Subtle expression alterations of multiple, IgA-relevant genes, collectively contribute to IgA deficiency in KS1 and RT1, but not in KS2. We propose that the joint study of MDEMs offers a principled approach for systematically mapping functional epigenetic variation in mammals.

scholarly journals p63 cooperates with CTCF to modulate chromatin architecture in skin keratinocytes

2019 ◽  
Author(s):  
Jieqiong Qu ◽  
Guoqiang Yi ◽  
Huiqing Zhou
Keyword(s):  
Transcription Factor ◽  
Three Dimensional ◽  
Chromatin Accessibility ◽  
Open Chromatin ◽  
Gene Promoters ◽  
Chromatin Architecture ◽  
Chromatin Interactions ◽  
Skin Keratinocytes ◽  
Eec Syndrome ◽  
Cell Type Specific

AbstractThe transcription factor p63 regulates epidermal genes and the enhancer landscape in skin keratinocytes. Its molecular function in controlling the chromatin structure is however not yet completely understood. Here we integrated multi-omics profiles, including the transcriptome, transcription factor DNA-binding and chromatin accessibility, in skin keratinocytes isolated from EEC syndrome patients carrying p63 mutations, to examine the role of p63 in shaping the chromatin architecture. We found decreased chromatin accessibility in p63-and CTCF-bound open chromatin regions that potentially contributed to gene deregulation in mutant keratinocytes. Cooperation of p63 and CTCF seemed to assist chromatin interactions between p63-bound enhancers and gene promoters in skin keratinocytes. Our study suggests an intriguing model where cell type-specific transcription factors such as p63 cooperate with the genome organizer CTCF in the three-dimensional chromatin space to regulate the transcription program important for the proper cell identity.

Human globin gene promoter sequences are sufficient for specific expression of a hybrid gene transfected into tissue culture cells

1987 ◽  
Vol 7 (1) ◽  
pp. 398-402
Author(s):  
T Rutherford ◽  
A W Nienhuis
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Gene Promoter ◽  
Gene Promoters ◽  
Globin Genes ◽  
Specific Expression ◽  
Tissue Specific ◽  
Promoter Sequences ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia

The contribution of the human globin gene promoters to tissue-specific transcription was studied by using globin promoters to transcribe the neo (G418 resistance) gene. After transfection into different cell types, neo gene expression was assayed by scoring colony formation in the presence of G418. In K562 human erythroleukemia cells, which express fetal and embryonic globin genes but not the adult beta-globin gene, the neo gene was expressed strongly from a fetal gamma- or embryonic zeta-globin gene promoter but only weakly from the beta promoter. In murine erythroleukemia cells which express the endogenous mouse beta genes, the neo gene was strongly expressed from both beta and gamma promoters. In two nonerythroid cell lines, human HeLa cells and mouse 3T3 fibroblasts, the globin gene promoters did not allow neo gene expression. Globin-neo genes were integrated in the erythroleukemia cell genomes mostly as a single copy per cell and were transcribed from the appropriate globin gene cap site. We conclude that globin gene promoter sequences extending from -373 to +48 base pairs (bp) (relative to the cap site) for the beta gene, -385 to +34 bp for the gamma gene, and -555 to +38 bp for the zeta gene are sufficient for tissue-specific and perhaps developmentally specific transcription.

scholarly journals Chromatin dynamics during hematopoiesis reveal discrete regulatory modules instructing differentiation

2020 ◽  
Author(s):  
Grigorios Georgolopoulos ◽  
Mineo Iwata ◽  
Nikoletta Psatha ◽  
Andrew Nishida ◽  
Tannishtha Som ◽  
...  
Keyword(s):  
Temporal Dynamics ◽  
Ex Vivo ◽  
Chromatin Accessibility ◽  
Dnase I ◽  
Lineage Commitment ◽  
Gene Promoters ◽  
Chromatin Dynamics ◽  
Regulatory Modules ◽  
Hypersensitive Sites ◽  
Regulatory Landscape

AbstractLineage commitment and differentiation is driven by the concerted action of master transcriptional regulators at their target chromatin sites. Multiple efforts have characterized the key transcription factors (TFs) that determine the various hematopoietic lineages. However, the temporal interactions between individual TFs and their chromatin targets during differentiation and how these interactions dictate lineage commitment remains poorly understood. We performed dense, daily, temporal profiling of chromatin accessibility (DNase I-seq) and gene expression changes (total RNA-seq) along ex vivo human erythropoiesis to comprehensively define developmentally regulated DNase I hypersensitive sites (DHSs) and transcripts. We link both distal DHSs to their target gene promoters and individual TFs to their target DHSs, revealing that the regulatory landscape is organized in distinct sequential regulatory modules that regulate lineage restriction and maturation. Finally, direct comparison of transcriptional dynamics (bulk and single-cell) and lineage potential between erythropoiesis and megakaryopoiesis illuminates the fine-scale temporal dynamics of these regulatory modules during lineage-resolution between these two fates. Collectively, these data provide novel insights into the global regulatory landscape during hematopoiesis.

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