HDAC Inhibitors As Novel Targeted Therapies for NUP98-HOXA9 AML Patients

Abstract Background: The chromosomal translocation t(7;11)(p15,p15) encodes the oncogenic transcription factor NUP98-HOXA9 which results in a fusion of the nucleoporin 98kDa (NUP98) and homeobox A9 (HOXA9) genes. The oncogenic mechanisms underlying this translocation remain poorly understood and patients are currently inadequately served by traditional cytotoxic chemotherapy regimens. Aims:To decipher the underlying biology of the NUP98-HOXA9 fusion protein and develop rational therapeutic strategies targeting its oncogenic mechanism. Methods: Human cellular models expressing NUP98-HOXA9, HOXA9 wt or NUP98 wt were established by retroviral transduction of HEK293FT human cell line and human hematopoietic progenitors (CD34+, hHP) isolated from donor cord blood. Chromatin immunoprecepitation experiments followed by sequencing (ChIP-seq) and quantitative ChIP (qChIP) were used to define fusion specific binding locations. Cloning regulatory regions of selected target genes in a luciferase vectorconfirmed the direct involvement of NUP98-HOXA9 in their regulation. RTQ-PCR and gene expression microarrays were used to evaluate expression levels. Co-Immunoprecipitation experiments validated protein-protein interactions and drug treatments were performed at IC50. Cell viability was analysed by apoptosis, proliferation and Colony Forming Unit assays. Results:Comparison of ChIP-seq data from HEK293FTmodels of NUP98-HOXA9, HOXA9 wt or NUP98 wt respectively, identified 4,471 target genomic regions of the fusion protein (FDR < 0.05), located within +4/-4 kb from the annotated Transcription Start Site (TSS) of 1,363 genes, with 399 genes common to HOXA9 wt and 5 to NUP98 wt. The NUP98-HOXA9 binding sites included enhancers of MEIS1, HOXA9 and PBX3 (PBX3 and HOXA9 were common to NUP98 wt and MEIS1 to HOXA9 wt). Together these transcription factors form a key activator complex that regulates the expression of genes involved in leukemogenesis and its overexpression is significant related to adverse prognosis in AML. Luciferase assays showed that the upregulation of this leukemic axis was directly induced by the interaction of NUP98-HOXA9 with the corresponding enhancer regions of MEIS1, HOXA9 and PBX3. Treatment of cells with HXR9, a specific peptide inhibitor of HOXA9 and PBX3 interaction, led to a selective decrease in the proliferation of hHP expressing NUP98-HOXA9, confirming the relevance of these target genes to its oncogenic mechanism. Combining ChIP-seq and gene expression data of three independent clones of hHP expressing NUP98-HOXA9 and patient samples (n = 5) harbouring t(7;11)(p15,p15) revealed a dual regulatory role of the fusion protein, in both repressing and activating target gene transcription where, for example, MEIS1, HOXA9, PBX3 and AFF3 were found overexpressed and BIRC3, SMAD1, FILIP1L and PTEN downregulated. Interactions of NUP98-HOXA9 with p300 and HDAC1 were shown to drive this transcriptional activation and repression, respectively. We found using qChIP experiments that p300 bound to the regulatory regions of the overexpressed genes only when NUP98-HOXA9 was present, whereas we observed significant enrichment of HDAC1 binding to the promoter regions of the downregulated genes when the fusion protein was expressed. Taking advantage of this latter observation, we demonstrated a dramatic inhibitory effect on the viability of hHP expressing NUP98-HOXA9after the treatment with subtherapeutic doses (IC50 = 4nM) of the HDAC inhibitor LBH-589 (Panobinostat) with no effect in control hHP transduced with an empty vector. Conclusion: An improved understanding of the pathobiology underlying recurrent translocation events in AML is a critical first step for the development of rational, targeted therapies. Here, we identify upregulation of the targetable MEIS1-HOXA9-PBX3 complex underpinning the leukemogenic activity of NUP98-HOXA9. Its activity in repressing transcription mediated through interaction with HDAC1, has been shown to be also a key pathogenic mechanism that can be exploited through use of HDAC inhibitors and potentially lead to a promising new therapy for this high-risk group of patients. Disclosures No relevant conflicts of interest to declare.

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Direct activation of Sex-lethal transcription by the Drosophila runt protein

Development ◽

10.1242/dev.126.1.191 ◽

1999 ◽

Vol 126 (1) ◽

pp. 191-200 ◽

Cited By ~ 3

Author(s):

S.G. Kramer ◽

T.M. Jinks ◽

P. Schedl ◽

J.P. Gergen

Keyword(s):

Transcriptional Activation ◽

Target Genes ◽

Specific Binding ◽

Binding Activity ◽

Transcriptional Activation Domain ◽

Downstream Target ◽

Early Promoter ◽

Dna Binding Activity ◽

Sex Lethal

Runt functions as a transcriptional regulator in multiple developmental pathways in Drosophila melanogaster. Recent evidence indicates that Runt represses the transcription of several downstream target genes in the segmentation pathway. Here we demonstrate that runt also functions to activate transcription. The initial expression of the female-specific sex-determining gene Sex-lethal in the blastoderm embryo requires runt activity. Consistent with a role as a direct activator, Runt shows sequence-specific binding to multiple sites in the Sex-lethal early promoter. Using an in vivo transient assay, we demonstrate that Runt's DNA-binding activity is essential for Sex-lethal activation in vivo. These experiments further reveal that increasing the dosage of runt alone is sufficient for triggering the transcriptional activation of Sex-lethal in males. In addition, a Runt fusion protein, containing a heterologous transcriptional activation domain activates Sex-lethal expression, indicating that this regulation is direct and not via repression of other repressors. Moreover, we demonstrate that a small segment of the Sex-lethal early promoter that contains Runt-binding sites mediates Runt-dependent transcriptional activation in vivo.

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Histone deacetylase 3 preferentially binds and collaborates with the transcription factor RUNX1 to repress AML1–ETO–dependent transcription in t(8;21) AML

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010707 ◽

2020 ◽

Vol 295 (13) ◽

pp. 4212-4223 ◽

Cited By ~ 1

Author(s):

Chun Guo ◽

Jian Li ◽

Nickolas Steinauer ◽

Madeline Wong ◽

Brent Wu ◽

...

Keyword(s):

Transcription Factor ◽

Fusion Protein ◽

Histone Deacetylase ◽

Transcriptional Activation ◽

Target Genes ◽

Chromosomal Translocation ◽

Histone Deacetylase 3 ◽

Genome Wide ◽

Related Transcription Factor ◽

Almost All

In up to 15% of acute myeloid leukemias (AMLs), a recurring chromosomal translocation, termed t(8;21), generates the AML1–eight–twenty-one (ETO) leukemia fusion protein, which contains the DNA-binding domain of Runt-related transcription factor 1 (RUNX1) and almost all of ETO. RUNX1 and the AML1–ETO fusion protein are coexpressed in t(8;21) AML cells and antagonize each other's gene-regulatory functions. AML1–ETO represses transcription of RUNX1 target genes by competitively displacing RUNX1 and recruiting corepressors such as histone deacetylase 3 (HDAC3). Recent studies have shown that AML1–ETO and RUNX1 co-occupy the binding sites of AML1–ETO–activated genes. How this joined binding allows RUNX1 to antagonize AML1–ETO–mediated transcriptional activation is unclear. Here we show that RUNX1 functions as a bona fide repressor of transcription activated by AML1–ETO. Mechanistically, we show that RUNX1 is a component of the HDAC3 corepressor complex and that HDAC3 preferentially binds to RUNX1 rather than to AML1–ETO in t(8;21) AML cells. Studying the regulation of interleukin-8 (IL8), a newly identified AML1–ETO–activated gene, we demonstrate that RUNX1 and HDAC3 collaboratively repress AML1–ETO–dependent transcription, a finding further supported by results of genome-wide analyses of AML1–ETO–activated genes. These and other results from the genome-wide studies also have important implications for the mechanistic understanding of gene-specific coactivator and corepressor functions across the AML1–ETO/RUNX1 cistrome.

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The TEA Transcription Factor Tec1 Confers Promoter-Specific Gene Regulation by Ste12-Dependent and -Independent Mechanisms

Eukaryotic Cell ◽

10.1128/ec.00251-09 ◽

2010 ◽

Vol 9 (4) ◽

pp. 514-531 ◽

Cited By ~ 20

Author(s):

Barbara Heise ◽

Julia van der Felden ◽

Sandra Kern ◽

Mario Malcher ◽

Stefan Brückner ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Transcriptional Activation ◽

Binding Sites ◽

Target Genes ◽

Specific Gene ◽

Dependent Manner ◽

A Genome ◽

Regulated Gene Expression

ABSTRACT In Saccharomyces cerevisiae, the TEA transcription factor Tec1 is known to regulate target genes together with a second transcription factor, Ste12. Tec1-Ste12 complexes can activate transcription through Tec1 binding sites (TCSs), which can be further combined with Ste12 binding sites (PREs) for cooperative DNA binding. However, previous studies have hinted that Tec1 might regulate transcription also without Ste12. Here, we show that in vivo, physiological amounts of Tec1 are sufficient to stimulate TCS-mediated gene expression and transcription of the FLO11 gene in the absence of Ste12. In vitro, Tec1 is able to bind TCS elements with high affinity and specificity without Ste12. Furthermore, Tec1 contains a C-terminal transcriptional activation domain that confers Ste12-independent activation of TCS-regulated gene expression. On a genome-wide scale, we identified 302 Tec1 target genes that constitute two distinct classes. A first class of 254 genes is regulated by Tec1 in a Ste12-dependent manner and is enriched for genes that are bound by Tec1 and Ste12 in vivo. In contrast, a second class of 48 genes can be regulated by Tec1 independently of Ste12 and is enriched for genes that are bound by the stress transcription factors Yap6, Nrg1, Cin5, Skn7, Hsf1, and Msn4. Finally, we find that combinatorial control by Tec1-Ste12 complexes stabilizes Tec1 against degradation. Our study suggests that Tec1 is able to regulate TCS-mediated gene expression by Ste12-dependent and Ste12-independent mechanisms that enable promoter-specific transcriptional control.

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Transcriptional Regulation of Erythropoiesis.

Blood ◽

10.1182/blood.v114.22.sci-7.sci-7 ◽

2009 ◽

Vol 114 (22) ◽

pp. SCI-7-SCI-7

Author(s):

Mitchell J. Weiss

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Transcriptional Activation ◽

Zinc Finger Protein ◽

Conflicts Of Interest ◽

Globin Gene ◽

Globin Genes ◽

Erythroid Development ◽

Globin Gene Expression

Abstract Abstract SCI-7 Efforts to define the mechanisms of globin gene expression and transcriptional control of erythrocyte formation have provided key insights into our understanding of developmental hematopoiesis. Our group has focused on GATA-1, a zinc finger protein that was initially identified through its ability to bind a conserved cis element that regulates globin gene expression. GATA-1 is essential for erythroid development and mutations in the GATA1 gene are associated with human cytopenias and leukemia. Several general principles have emerged through studies to define the mechanisms of GATA-1 action. First, GATA-1 activates not only globin genes, but also virtually every gene that defines the erythroid phenotype. This observation sparked successful gene discovery efforts to identify new components of erythroid development and physiology. Second, GATA-1 also represses transcription through multiple mechanisms. This property may help to explain how GATA-1 regulates hematopoietic lineage commitment and also how GATA1 mutations contribute to cancer, since several directly repressed targets are proto-oncogenes. Third, GATA-1 regulates not only protein coding genes, but also microRNAs, which in turn, modulate erythropoiesis through post-transcriptional mechanisms. Fourth, GATA-1 interacts with other essential erythroid-specific and ubiquitous transcription factors. These protein interactions regulate gene expression by influencing chromatin modifications and controlling three-dimensional proximity between widely spaced DNA elements. Recently, we have combined transcriptome analysis with ChIP-chip and ChIP-seq studies to correlate in vivo occupancy of DNA by GATA-1 and other transcription factors with mRNA expression genome-wide in erythroid cells. These studies better elucidate how GATA-1 recognizes DNA, discriminates between transcriptional activation versus repression and interacts functionally with other nuclear proteins. I will review published and new aspects of our work in these areas. Disclosures No relevant conflicts of interest to declare.

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CBX8, a Polycomb-Group Protein, Is Essential for MLL-AF9-Induced Leukemogenesis

Blood ◽

10.1182/blood.v116.21.4174.4174 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4174-4174

Author(s):

Jiaying Tan ◽

Jay L. Hess

Keyword(s):

Gene Expression ◽

Cell Growth ◽

Cell Lines ◽

Transcriptional Activation ◽

Fusion Proteins ◽

Conflicts Of Interest ◽

Leukemia Cell ◽

Polycomb Group ◽

Human Leukemia ◽

Interaction Sites

Abstract Abstract 4174 Trithorax and Polycomb-group (Trx-G and Pc-G) proteins are antagonistic regulators of homeobox-containing (Hox) gene expression that play a major role in regulation of hematopoiesis and leukemogenesis. Mixed lineage leukemia (MLL), a mammalian Trx-G protein, is a histone methyltransferase crucial for embryonic development and hematopoiesis that is commonly altered by translocation in acute leukemia. Recent evidence suggests that transformation by MLL fusion proteins is dependent on multiple interaction complexes, including the polymerase associated factor complex (PAFc) and the elongation activating protein complex (EAPc) or a closely related AF4 family/ENL family/P-TEFb complex (AEPc). CBX8 is a human PcG protein, functioning as a transcription repressor in the polycomb repressive complex 1 (PRC1). Previous studies have shown that CBX8 also interacts with the EAPc components AF9 and ENL; however, its role in leukemogenesis is unknown. To elucidate the significance of this interaction between these two proteins thought to have antagonistic function, we generated a large series of point mutations in AF9 and identified two amino acids that are essential for CBX8 interaction but preserve the interaction with other EAP components. Mutation of the two sites reduced the transcriptional activation of the MLL-AF9 target promoters by nearly 50% and completely inhibits the ability of MLL-AF9 to immortalize bone marrow (BM) as assessed by methylcellulose replating assays. This finding suggests that CBX8 interaction is essential for MLL-AF9-induced leukemogenesis. Several lines of evidence further support this finding. First, CBX8 knockdown by siRNAs decreased MLL-AF9-induced transcriptional activation by approximately 50%. Second, the ability of MLL-AF9 to transform primary BM was markedly reduced by retroviral shCbx8 transduction. Notably, this inhibitory effect is specific for MLL-AF9 because the BM transformation ability of E2A-HLF was unaffected by Cbx8 suppression. Third, Cbx8 suppression by shCbx8 in MLL-AF9 and MLL-ENL, but not E2A-HLF transformed AML cell lines, significantly inhibited the expression of MLL-dependent target genes, as well as cell growth and colony forming ability. Fourth, inducing CBX8 knockdown in human leukemia cell lines expressing MLL-AF9 led to a marked decrease in the localization of basic transcription machinery at the Hoxa9 locus and a corresponding reduction in Hoxa9 transcription. Importantly, the observed effects of CBX8 on MLL-rearranged leukemia cells are PRC1-independent: no effects on MLL target gene expression, cell growth, or BM transformation ability were observed by suppressing other core components of PRC1. Taken together, our results indicate that CBX8, independent of its transcription repression role in PRC1, interacts with and synergizes with MLL fusion proteins to promote leukemogenesis. Defining the interaction sites between AF9/ENL and CBX8 and the dependence of other AML subtypes and normal hematopoiesis on CBX8 will be important for the further development of agents that target this mechanism in MLL-rearranged and potentially other AML subtypes. Disclosures: No relevant conflicts of interest to declare.

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Early Different Downstream Target of Glucocorticoid Receptor Contributing to Glucocorticoids Sensitivity in Kasumi-1 Cells

Blood ◽

10.1182/blood.v128.22.5132.5132 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5132-5132

Author(s):

Wenbin Gu ◽

Meng Li ◽

Liang Liang ◽

Jian Zhang ◽

Chongye Guo ◽

...

Keyword(s):

Glucocorticoid Receptor ◽

Fusion Protein ◽

Cell Line ◽

Myeloid Leukemia ◽

Target Genes ◽

Conflicts Of Interest ◽

Critical Role ◽

Leukemia Cell ◽

Leukemia Cells ◽

Non Coding Rnas

Abstract The t(8;21) chromosome translocation frequently occurs in acute myeloid leukemia (AML), resulting in an in-frame fusion between the DNA-binding domain of AML1 and almost the entire of ETO gene. The fusion AML1-ETO protein is thought to play a critical role in the abnormal proliferation and differentiation of myeloid leukemia cells, such as Kasumi-1 and SKNO-1 cells. Glucocorticoids (GC) can induce apoptosis in these cells at low concentrations, whereas most other myeloid leukemia cell lines are resistant to glucocorticoid-induced apoptosis. To experimentally address possible sensitive mechanisms in leukemia cells with AML1-ETO translocation, we generated aGC-resistant Kasumi-1 cell line by induction of 10-6 M dexamethasone (Dex) for three weeks. The IC50 of Dex to cells is increased from 2.5×10-8 M for original GC-sensitive Kasumi-1 cell line ( K-S cell line) to more than 1×10-5 M for induced GC-resistant Kasumi-1 cell line (K-R cell line). Since GC resistance often results from mutations in the glucocorticoid receptor (GR), all the exons of GR gene were sequenced and no mutation was found in K-R cells. Comparing to those in K-S cells, the GR protein level didn't decrease in K-R cells after 2h, 4h, 8h, 12h and 24h exposure to dexamethasone. Given that the difference of direct GR downstream genes between K-S and K-R cells may play a key role in the GC sensitivity, we systematically analyzed the changes of gene expression induced by Dex versus ethanol vehicle for 8h in K-S and K-R cells by high throughput RNA sequencing. The time point of 8h was selected according to the expression peaks of several foregone GR target genes after Dex induction. There were found 32 genes conversely regulated in K-S and K-R cells, including 14 mRNAs and 18 long non-coding RNAs. Pathway analysis indicated that the upregulated genes in K-S cells might promote the AML1-ETO fusion protein degradation by proteasomes, while the component genes of this pathway were downregulated in K-R cells. Further validation and function studies of these mRNAs and long non-coding RNAs are ongoing. Our data suggested that the downstream targets of GR among GC-sensitive and -resistant Kasumi-1 cells were significant different and they may contribute to the GC sensitivity and resistance by degradation or reservation of AML-ETO fusion protein and the regulation of apoptosis in t(8;21) leukemia cell subtype. Disclosures No relevant conflicts of interest to declare.

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Purification and characterization of the stage-specific embryonic enhancer-binding protein SSAP-1.

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1746 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1746-1758 ◽

Cited By ~ 10

Author(s):

D J DeAngelo ◽

J DeFalco ◽

G Childs

Keyword(s):

Gene Expression ◽

Molecular Weight ◽

Transcriptional Activation ◽

Target Genes ◽

Gene Activation ◽

Upstream Sequence ◽

Blastula Stage ◽

Sequence Element ◽

Enhancer Element ◽

Beta Gene

We have demonstrated that a highly conserved segment of DNA between positions -288 and -317 (upstream sequence element IV [USE IV]) is largely responsible for the transcriptional activation of the sea urchin H1-beta histone gene during the blastula stage of embryogenesis. This sequence is capable of acting as an embryonic enhancer element, activating target genes in a stage-specific manner. Nuclear extracts prepared from developmentally-staged organisms before and after the gene is activated all contain a factor which specifically binds to the enhancer. We have purified a 43-kDa polypeptide which binds to and footprints the USE IV enhancer element. We refer to this protein as stage-specific activator protein 1 (SSAP-1). Early in development before the enhancer is active, SSAP appears as a 43-kDa monomer, but it undergoes a change in its molecular weight beginning at about 12 h postfertilization (early blastula) which precisely parallels the increase in H1-beta gene expression. Modified SSAP has an apparent molecular mass of approximately 90 to 100 kDa and contains at least one 43-kDa SSAP polypeptide. Thus, it is the disappearance of the 43-kDa species and the appearance of the 90- to 100-kDa species which coincide with the H1-beta gene activation. The correlation between the change in molecular weight of SSAP and the stage-specific activation of H1-beta gene expression strongly suggests that this higher-molecular-weight form of SSAP is directly responsible for the blastula stage-specific transcriptional activation of the late H1 gene.

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Histone deacetylase inhibitors suppress IL-2–mediated gene expression prior to induction of apoptosis

Blood ◽

10.1182/blood.v96.4.1490.h8001490_1490_1495 ◽

2000 ◽

Vol 96 (4) ◽

pp. 1490-1495 ◽

Cited By ~ 3

Author(s):

Yuko Koyama ◽

Masaaki Adachi ◽

Masuo Sekiya ◽

Mutsuhiro Takekawa ◽

Kohzoh Imai

Keyword(s):

Gene Expression ◽

Histone Deacetylase ◽

Transcriptional Activation ◽

Histone Deacetylase Inhibitors ◽

Trichostatin A ◽

Hdac Inhibitors ◽

K562 Cells ◽

Histone Hyperacetylation ◽

Induced Apoptosis ◽

Do So

Histone deacetylase (HDAC) inhibitors can induce transcriptional activation of a number of genes and induce cellular differentiation as histone acetylation levels increase. Although these inhibitors induce apoptosis in several cell lines, the precise mechanism by which they do so remains obscure. This study shows that HDAC inhibitors, sodium butyrate and trichostatin A (TSA), abrogate interleukin (IL)-2–mediated gene expression in IL-2–dependent cells. The HDAC inhibitors readily induced apoptosis in IL-2–dependent ILT-Mat cells and BAF-B03 transfectants expressing the IL-2 receptor βc chain, whereas they induced far less apoptosis in cytokine-independent K562 cells. However, these inhibitors similarly increased acetylation levels of histones in both cells. Although histone hyperacetylation is believed to lead to transcriptional activation, the results showed an abrogation of IL-2–mediated induction of c-myc,bag-1, and LC-PTP gene expression. This observed abrogation of gene expression occurred prior to phosphatidylserine externalization, a process that occurs in early apoptotic cells. Considering the biologic role played by IL-2–mediated gene expression in cell survival, these data suggest that its abrogation may contribute to the apoptotic process induced by HDAC inhibitors.

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Candidate cancer driver mutations in superenhancers and long-range chromatin interaction networks

10.1101/236802 ◽

2017 ◽

Cited By ~ 4

Author(s):

Lina Wadi ◽

Liis Uusküla-Reimand ◽

Keren Isaev ◽

Shimin Shuai ◽

Vincent Huang ◽

...

Keyword(s):

Gene Expression ◽

Long Range ◽

Target Genes ◽

Tumor Biology ◽

Regulatory Elements ◽

Chromatin Interaction ◽

Driver Mutations ◽

Regulatory Regions ◽

Protein Coding ◽

Primary Tumors

AbstractA comprehensive catalogue of the mutations that drive tumorigenesis and progression is essential to understanding tumor biology and developing therapies. Protein-coding driver mutations have been well-characterized by large exome-sequencing studies, however many tumors have no mutations in protein-coding driver genes. Non-coding mutations are thought to explain many of these cases, however few non-coding drivers besides TERT promoter are known. To fill this gap, we analyzed 150,000 cis-regulatory regions in 1,844 whole cancer genomes from the ICGC-TCGA PCAWG project. Using our new method, ActiveDriverWGS, we found 41 frequently mutated regulatory elements (FMREs) enriched in non-coding SNVs and indels (FDR<0.05) characterized by aging-associated mutation signatures and frequent structural variants. Most FMREs are distal from genes, reported here for the first time and also recovered by additional driver discovery methods. FMREs were enriched in super-enhancers, H3K27ac enhancer marks of primary tumors and long-range chromatin interactions, suggesting that the mutations drive cancer by distally controlling gene expression through threedimensional genome organization. In support of this hypothesis, the chromatin interaction network of FMREs and target genes revealed associations of mutations and differential gene expression of known and novel cancer genes (e.g., CNNB1IP1, RCC1), activation of immune response pathways and altered enhancer marks. Thus distal genomic regions may include additional, infrequently mutated drivers that act on target genes via chromatin loops. Our study is an important step towards finding such regulatory regions and deciphering the somatic mutation landscape of the non-coding genome.

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The Drosophila HP1 family is associated with active gene expression across chromatin contexts

Genetics ◽

10.1093/genetics/iyab090 ◽

2021 ◽

Author(s):

John M Schoelz ◽

Justina X Feng ◽

Nicole C Riddle

Keyword(s):

Gene Expression ◽

Protein Interactions ◽

Transcriptional Activation ◽

Target Genes ◽

Family Members ◽

Transcription Start ◽

Heterochromatin Formation ◽

Transcription Start Sites ◽

Polymerase Pausing ◽

Hp1 Proteins

Abstract Drosophila Heterochromatin Protein 1a (HP1a) is essential for heterochromatin formation and is involved in transcriptional silencing. However, certain loci require HP1a to be transcribed. One model posits that HP1a acts as a transcriptional silencer within euchromatin while acting as an activator within heterochromatin. However, HP1a has been observed as an activator of a set of euchromatic genes. Therefore, it is not clear whether, or how, chromatin context informs the function of HP1 proteins. To understand the role of HP1 proteins in transcription, we examined the genome-wide binding profile of HP1a as well as two other Drosophila HP1 family members, HP1B and HP1C, to determine whether coordinated binding of these proteins is associated with specific transcriptional outcomes. We found that HP1 proteins share many of their endogenous binding targets. These genes are marked by active histone modifications and are expressed at higher levels than non-target genes in both heterochromatin and euchromatin. In addition, HP1 binding targets displayed increased RNA polymerase pausing compared to non-target genes. Specifically, co-localization of HP1B and HP1C was associated with the highest levels of polymerase pausing and gene expression. Analysis of HP1 null mutants suggests these proteins coordinate activity at transcription start sites to regulate transcription. Depletion of HP1B or HP1C alters expression of protein-coding genes bound by HP1 family members. Our data broaden understanding of the mechanism of transcriptional activation by HP1a and highlight the need to consider particular protein-protein interactions, rather than broader chromatin context, to predict impacts of HP1 at transcription start sites.

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