PUBLICATIONS
First/Senior-author papers
Please see my Google Scholar for the latest list
MAPPING MAMMALIAN 3D GENOMES BY MICRO-C
Slobodyanyuk, E., Cattoglio, C., Hsieh T.H.S. (2022) Methods Mol Biol
3D genome mapping aims at connecting the physics of chromatin folding to the underlying biological events, and applications of various chromosomal conformation capture (3C) assays continue to discover critical roles of genome folding in regulating nuclear functions. To interrogate the full spectrum of chromatin folding ranging from the level of nucleosomes to full chromosomes in mammals, we developed an enhanced 3C-based method called Micro-C. The protocol employs Micrococcal nuclease (MNase) to fragment the genome, which overcomes the resolution limit of restriction enzyme-based methods, enabling the estimation of contact frequencies between proximal nucleosomes. Such improvements successfully resolve the fine-scale level of chromatin folding, including enhancer-promoter or promoter-promoter interactions, genic and nucleosomal folding, and boost the signal-to-noise ratio in detecting loops and substructures underlying TADs. In this chapter, we will thoroughly discuss the details of the Micro-C protocol and critical parameters to consider for generating high-quality Micro-C maps.
COHESIN RESIDENCY DETERMINES CHROMATIN LOOP PATTERNS
Costantino, L.*, Hsieh, T.H.S.*, Lamothe, R.*, Darzacq, X., and Koshland, D. (2020) eLIFE
The organization of chromatin into higher order structures is essential for chromosome segregation, the repair of DNA-damage, and the regulation of gene expression. Using Micro-C XL to detect chromosomal interactions, we observed the pervasive presence of cohesin-dependent loops with defined positions throughout the genome of budding yeast, as seen in mammalian cells. In early S phase, cohesin stably binds to cohesin associated regions (CARs) genome-wide. Subsequently, positioned loops accumulate with CARs at the bases of the loops. Cohesin regulators Wpl1 and Pds5 alter the levels and distribution of cohesin at CARs, changing the pattern of positioned loops. From these observations, we propose that cohesin with loop extrusion activity is stopped by preexisting CAR-bound cohesins, generating positioned loops. The patterns of loops observed in a population of wild-type and mutant cells can be explained by this mechanism, coupled with a heterogeneous residency of cohesin at CARs in individual cells.
RESOLVING THE 3D LANDSCAPE OF TRANSCRIPTION-LINKED MAMMALIAN CHROMATIN FOLDING
Hsieh, T.H.S., Cattoglio, C., Slobodyanyuk, E., Hansen, A.S., Rando, O.J., Tjian, R., and Darzacq, X. (2020). Molecular Cell
Whereas folding of genomes at the large scale of epigenomic compartments and topologically associating domains (TADs) is now relatively well understood, how chromatin is folded at finer scales remains largely unexplored in mammals. Here, we overcome some limitations of conventional 3C-based methods by using high-resolution Micro-C to probe links between 3D genome organization and transcriptional regulation in mouse stem cells. Combinatorial binding of transcription factors, cofactors, and chromatin modifiers spatially segregates TAD regions into various finer-scale structures with distinct regulatory features including stripes, dots, and domains linking promoters-to-promoters (P-P) or enhancers-to-promoters (E-P) and bundle contacts between Polycomb regions. E-P stripes extending from the edge of domains predominantly link co-expressed loci, often in the absence of CTCF and cohesin occupancy. Acute inhibition of transcription disrupts these gene-related folding features without altering higher-order chromatin structures. Our study uncovers previously obscured finer-scale genome organization, establishing functional links between chromatin folding and gene regulation.
DISTINCT CLASSES OF CHROMATIN LOOPS REVEALED BY DELETION OF AN RNA-BINDING REGION IN CTCF
Hansen, A.S.*, Hsieh, T.H.S.*, Cattoglio, C.*, Pustova, I., Saldaña-Meyer, R., Reinberg, D., Darzacq, X., and Tjian, R. (2019) Molecular Cell
Mammalian genomes are folded into topologically associating domains (TADs), consisting of chromatin loops anchored by CTCF and cohesin. Some loops are cell-type specific. Here we asked whether CTCF loops are established by a universal or locus-specific mechanism. Investigating the molecular determinants of CTCF clustering, we found that CTCF self-association in vitro is RNase sensitive and that an internal RNA-binding region (RBRi) mediates CTCF clustering and RNA interaction in vivo. Strikingly, deleting the RBRi impairs about half of all chromatin loops in mESCs and causes deregulation of gene expression. Disrupted loop formation correlates with diminished clustering and chromatin binding of RBRi mutant CTCF, which in turn results in a failure to halt cohesin-mediated extrusion. Thus, CTCF loops fall into at least two classes: RBRi-independent and RBRi-dependent loops. We speculate that evidence for RBRi-dependent loops may provide a molecular mechanism for establishing cell-specific CTCF loops, potentially regulated by RNA(s) or other RBRi-interacting partners.
TRANSFER RNA GENES AFFECT CHROMOSOME STRUCTURE AND FUNCTION VIA LOCAL EFFECTS
Hamdani, O.*, Dhillon, N.*, Hsieh, T.H.S.*, Fujita, T.*, Ocampo, J., Kirkland, J.G., Lawrimore, J., Kobayashi, T.J., Friedman, B., Fulton, D., et al. (2019) Mol Cell Biol
The genome is packaged and organized in an ordered, nonrandom manner, and specific chromatin segments contact nuclear substructures to mediate this organization. tRNA genes (tDNAs) are binding sites for transcription factors and architectural proteins and are thought to play an important role in the organization of the genome. In this study, we investigate the roles of tDNAs in genomic organization and chromosome function by editing a chromosome so that it lacked any tDNAs. Surprisingly our analyses of this tDNA-less chromosome show that loss of tDNAs does not grossly affect chromatin architecture or chromosome tethering and mobility. However, loss of tDNAs affects local nucleosome positioning and the binding of SMC proteins at these loci. The absence of tDNAs also leads to changes in centromere clustering and a reduction in the frequency of long-range HML-HMR heterochromatin clustering with concomitant effects on gene silencing. We propose that the tDNAs primarily affect local chromatin structure, which results in effects on long-range chromosome architecture.
MICRO-C XL: ASSAYING CHROMOSOME CONFORMATION FROM THE NUCLEOSOME TO THE ENTIRE GENOME
Hsieh, T.H.S., Fudenberg, G., Goloborodko, A., and Rando, O.J. (2016) Nature Methods
We present Micro-C XL, an improved method for analysis of chromosome folding at mononucleosome resolution. Using long crosslinkers and isolation of insoluble chromatin, Micro-C XL increases signal-to-noise ratio. Micro-C XL maps of budding and fission yeast genomes capture both short-range chromosome fiber features such as chromosomally interacting domains and higher order features such as centromere clustering. Micro-C XL provides a single assay to interrogate chromosome folding at length scales from the nucleosome to the full genome.
MAPPING NUCLEOSOME RESOLUTION CHROMOSOME FOLDING IN YEAST BY MICRO-C
Hsieh, T.H.S., Weiner, A., Lajoie, B., Dekker, J., Friedman, N., and Rando, O.J. (2015) Cell
We describe a Hi-C-based method, Micro-C, in which micrococcal nuclease is used instead of restriction enzymes to fragment chromatin, enabling nucleosome resolution chromosome folding maps. Analysis of Micro-C maps for budding yeast reveals abundant self-associating domains similar to those reported in other species, but not previously observed in yeast. These structures, far shorter than topologically associating domains in mammals, typically encompass one to five genes in yeast. Strong boundaries between self-associating domains occur at promoters of highly transcribed genes and regions of rapid histone turnover that are typically bound by the RSC chromatin-remodeling complex. Investigation of chromosome folding in mutants confirms roles for RSC, “gene looping” factor Ssu72, Mediator, H3K56 acetyltransferase Rtt109, and the N-terminal tail of H4 in folding of the yeast genome. This approach provides detailed structural maps of a eukaryotic genome, and our findings provide insights into the machinery underlying chromosome compaction.
HIGH-RESOLUTION CHROMATIN DYNAMICS DURING A YEAST STRESS RESPONSE
Weiner, A.*, Hsieh, T.H.S.*, Appleboim, A.*, Chen, H.V., Rahat, A., Amit, I., Rando, O.J., and Friedman, N. (2015) Molecular Cell
Covalent histone modifications are highly conserved and play multiple roles in eukaryotic transcription regulation. Here, we mapped 26 histone modifications genome-wide in exponentially growing yeast and during a dramatic transcriptional reprogramming—the response to diamide stress. We extend prior studies showing that steady-state histone modification patterns reflect genomic processes, especially transcription, and display limited combinatorial complexity. Interestingly, during the stress response we document a modest increase in the combinatorial complexity of histone modification space, resulting from roughly 3% of all nucleosomes transiently populating rare histone modification states. Most of these rare histone states result from differences in the kinetics of histone modification that transiently uncouple highly correlated marks, with slow histone methylation changes often lagging behind the more rapid acetylation changes. Explicit analysis of modification dynamics uncovers ordered sequences of events in gene activation and repression. Together, our results provide a comprehensive view of chromatin dynamics during a massive transcriptional upheaval.
PRE-PRINTS
ENHANCER-PROMOTER INTERACTIONS AND TRANSCRIPTION ARE MAINTAINED UPON ACUTE LOSS OF CTCF, COHESIN, WAPL, AND YY1
Hsieh, T.H.S., Cattoglio, C., Slobodyanyuk, E., Hansen, A.S., Darzacq, X., and Tjian, R. (2021)
It remains unclear why acute depletion of CTCF and cohesin only marginally affects expression of most genes despite substantially perturbing 3D genome folding at the level of domains and structural loops. To address this conundrum, we used high-resolution Micro-C and nascent transcript profiling to find that enhancer-promoter (E-P) interactions are largely insensitive to acute (3-hour) depletion of CTCF, cohesin, and WAPL. YY1 has been proposed to be a structural regulator of E-P loops, but acute YY1 depletion also had minimal effects on E-P loops, transcription, and 3D genome folding. Strikingly, live-cell single-molecule imaging revealed that cohesin depletion reduced transcription factor binding to chromatin. Thus, although neither CTCF, cohesin, WAPL, nor YY1 are required for the short-term maintenance of most E-P interactions and gene expression, we propose that cohesin may serve as a “transcription factor binding platform” that facilitates transcription factor binding to chromatin.
COHESIN RESIDENCY DETERMINES CHROMATIN LOOP PATTERNS
Costantino, L.*, Hsieh, T.H.S.*, Lamothe, R., Darzacq, X., and Koshland, D. (2020)
The organization of chromatin into higher-order structures is essential for chromosome segregation, the repair of DNA-damage, and the regulation of gene expression. Using Micro-C XL to detect chromosomal interactions, we observed the pervasive presence of cohesin-dependent loops with defined positions throughout the genome of budding yeast, as seen in mammalian cells. In early S phase, cohesin stably binds to cohesin associated regions (CARs) genome-wide. Subsequently, positioned loops accumulate with CARs at the bases of the loops. Cohesin regulators Wpl1 and Pds5 alter the levels and distribution of cohesin at CARs, changing the pattern of positioned loops. From these observations, we propose that cohesin with loop extrusion activity is stopped by preexisting CAR-bound cohesins, generating positioned loops. The patterns of loops observed in a population of wild-type and mutant cells can be explained by this mechanism, coupled with a heterogeneous residency of cohesin at CARs in individual cells.
RESOLVING THE 3D LANDSCAPE OF TRANSCRIPTION-LINKED MAMMALIAN CHROMATIN FOLDING
Hsieh, T.H.S., Slobodyanyuk, E., Cattoglio, C., Hansen, A.S., Rando, O.J., Tjian, R., and Darzacq, X. (2019).
Chromatin folding below the scale of topologically associating domains (TADs) remains largely unexplored in mammals. Here, we used a high-resolution 3C-based method, Micro-C, to probe links between 3D-genome organization and transcriptional regulation in mouse stem cells. Combinatorial binding of transcription factors, cofactors, and chromatin modifiers spatially segregate TAD regions into “microTADs” with distinct regulatory features. Enhancer-promoter and promoter-promoter interactions extending from the edge of these domains predominantly link co-regulated loci, often independently of CTCF/Cohesin. Acute inhibition of transcription disrupts the gene-related folding features without altering higher-order chromatin structures. Intriguingly, we detect “two-start” zig-zag 30-nanometer chromatin fibers. Our work uncovers the finer-scale genome organization that establishes novel functional links between chromatin folding and gene regulation.
ULTRASTRUCTURAL DETAILS OF MAMMALIAN CHROMOSOME ARCHITECTURE
Krietenstein, N., Abraham, S., Venev, S. V., Abdennur, N., Gibcus, J., Hsieh, T.H.S., Parsi, K.M., Yang, L., Maehr, R., Mirny, L.A., et al. (2020)
Over the past decade, 3C-related methods, complemented by increasingly detailed microscopic views of the nucleus, have provided unprecedented insights into chromosome folding in vivo. Here, to overcome the resolution limits inherent to the majority of genome-wide chromosome architecture mapping studies, we extend a recently-developed Hi-C variant, Micro-C, to map chromosome architecture at nucleosome resolution in human embryonic stem cells and fibroblasts. Micro-C maps robustly capture well-described features of mammalian chromosome folding including A/B compartment organization, topologically associating domains (TADs), and cis interaction peaks anchored at CTCF binding sites, while also providing a detailed 1-dimensional map of nucleosome positioning and phasing genome-wide. Compared to high-resolution in situ Hi-C, Micro-C exhibits substantially improved signal-to-noise with an order of magnitude greater dynamic range, enabling not only localization of domain boundaries with single-nucleosome accuracy, but also resolving more than 20,000 additional looping interaction peaks in each cell type. Intriguingly, many of these newly-identified peaks are localized along stripe patterns and form transitive grids, consistent with their anchors being pause sites impeding the process of cohesin-dependent loop extrusion. Together, our analyses provide the highest resolution maps of chromosome folding in human cells to date, and provide a valuable resource for studies of chromosome folding mechanisms.
AN RNA-BINDING REGION REGULATES CTCF CLUSTERING AND CHROMATIN LOOPING
Hansen, A.S.*, Hsieh, T.H.S.*, Cattoglio, C.*, Pustova, I., Darzacq, X., and Tjian, R. (2018)
Mammalian genomes are folded into Topologically Associating Domains (TADs), consisting of cell-type specific chromatin loops anchored by CTCF and cohesin. Since CTCF and cohesin are expressed ubiquitously, how cell-type specific CTCF-mediated loops are formed poses a paradox. Here we show RNase-sensitive CTCF self-association in vitro and that an RNA-binding region (RBR) mediates CTCF clustering in vivo. Intriguingly, deleting the RBR abolishes or impairs almost half of all chromatin loops in mouse embryonic stem cells. Disrupted loop formation correlates with abrogated clustering and diminished chromatin binding of the RBR mutant CTCF protein, which in turn results in a failure to halt cohesin-mediated extrusion. Thus, CTCF loops fall into at least 2 classes: RBR-independent and RBR-dependent loops. We suggest that evidence for distinct classes of RBR-dependent loops may provide a mechanism for establishing cell-specific CTCF loops regulated by RNAs and other RBR partner.
TRANSFER RNA GENES AFFECT CHROMOSOME STRUCTURE AND FUNCTION VIA LOCAL EFFECTS
Hamdani, O.*, Dhillon, N.*, Hsieh, T.H.S.*, Fujita, T.*, Ocampo, J., Kirkland, J.G., Lawrimore, J., Kobayashi, T.J., Friedman, B., Fulton, D., et al. (2018)
The genome is packaged and organized in an ordered, non-random manner and specific chromatin segments contact nuclear substructures to mediate this organization. While transfer RNA genes (tDNAs) are essential for the generation of tRNAs, these loci are also binding sites for transcription factors and architectural proteins and are thought to play an important role in the organization of the genome. In this study, we investigate the role of tDNAs in genomic organization and chromosome function by editing a chromosome so that it lacks any tDNAs. Surprisingly our analyses of this tDNA-less chromosome show that loss of tDNAs does not grossly affect chromosome folding or chromosome tethering. However, loss of tDNAs affects local nucleosome positioning and the binding of SMC proteins at these loci. The absence of tDNAs also leads to changes in centromere clustering and a reduction in the frequency of long range HML-HMR heterochromatin clustering. We propose that the tDNAs primarily affect local chromatin structure that result in effects on long-range chromosome architecture.
MICRO-C XL: ASSAYING CHROMOSOME CONFORMATION AT LENGTH SCALES FROM THE NUCLEOSOME TO THE ENTIRE GENOME
Hsieh, T.H.S., Fudenberg, G., Goloborodko, A., and Rando, O.J. (2016)
Structural analysis of chromosome folding in vivo has been revolutionized by Chromosome Conformation Capture (3C) and related methods, which use proximity ligation to identify chromosomal loci in physical contact. We recently described a variant 3C technique, Micro-C, in which chromatin is fragmented to mononucleosomes using micrococcal nuclease, enabling nucleosome-resolution folding maps of the genome. Here, we describe an improved Micro-C protocol using long crosslinkers, termed Micro-C XL, which exhibits greatly increased signal to noise, and provides further insight into the folding of the yeast genome. We also find that signal to noise is much improved in Micro-C XL libraries generated from relatively insoluble chromatin as opposed to soluble material, providing a simple method to physically enrich for bona-fide long-range interactions. Micro-C XL maps of the budding and fission yeast genomes reveal both short-range chromosome fiber features such as chromosomally-interacting domains (CIDs), as well as higher-order features such as clustering of centromeres and telomeres, thereby addressing the primary discrepancy between prior Micro-C data and reported 3C and Hi-C analyses. Interestingly, comparison of chromosome folding maps of S. cerevisiae and S. pombe revealed widespread qualitative similarities, yet quantitative differences, between these distantly-related species. Micro-C XL thus provides a single assay suitable for interrogation of chromosome folding at length scales from the nucleosome to the full genome.
CONDENSIN-DEPENDENT CHROMATIN CONDENSATION REPRESSES TRANSCRIPTION GLOBALLY DURING QUIESCENCE
Swygert, S.G., Kim, S., Wu, X., Fu, T., Hsieh, T.H.S., Rando, O.J., Eisenman, R.N., Shendure, J., McKnight, J.N., and Tsukiyama, T. (2018)
Quiescence is a stress-resistant state in which cells reversibly exit the mitotic cell cycle and suspend most cellular processes. Quiescence is essential for stem cell maintenance and its misregulation is implicated in tumor formation. One of the conserved hallmarks of quiescent cells, from Saccharomyces cerevisiae to humans, is highly condensed chromatin. Here, we use Micro-C XL to map chromatin contacts at single-nucleosome resolution genome-wide to elucidate mechanisms and functions of condensed chromatin in quiescent S. cerevisiae cells. We describe previously uncharacterized chromatin domains on the order of 10-60 kilobases that in quiescent cells are formed by condensin-mediated chromatin loops. Conditional depletion of condensin prevents chromatin condensation during quiescence entry and leads to widespread transcriptional de-repression. We further demonstrate that condensin-dependent chromatin compaction is conserved in quiescent human fibroblasts. We propose that condensin-dependent condensation of chromatin represses transcription throughout the quiescent cell genome.
PUBLICATIONS
Collaborative papers
ULTRASTRUCTURAL DETAILS OF MAMMALIAN CHROMOSOME ARCHITECTURE
Krietenstein, N., Abraham, S., Venev, S. V., Abdennur, N., Gibcus, J., Hsieh, T.H.S., Parsi, K.M., Yang, L., Maehr, R., Mirny, L.A., et al. (2020) Molecular Cell
Over the past decade, 3C-related methods have provided remarkable insights into chromosome folding in vivo. To overcome the limited resolution of prior studies, we extend a recently developed Hi-C variant, Micro-C, to map chromosome architecture at nucleosome resolution in human ESCs and fibroblasts. Micro-C robustly captures known features of chromosome folding including compartment organization, topologically associating domains, and interactions between CTCF binding sites. In addition, Micro-C provides a detailed map of nucleosome positions and localizes contact domain boundaries with nucleosomal precision. Compared to Hi-C, Micro-C exhibits an order of magnitude greater dynamic range, allowing the identification of ∼20,000 additional loops in each cell type. Many newly identified peaks are localized along extrusion stripes and form transitive grids, consistent with their anchors being pause sites impeding cohesin-dependent loop extrusion. Our analyses comprise the highest-resolution maps of chromosome folding in human cells to date, providing a valuable resource for studies of chromosome organization.
3D ATAC-PALM: SUPER-RESOLUTION IMAGING OF THE ACCESSIBLE GENOME
Xie, L., Dong, P., Chen, X., Hsieh, T.H.S., Banala, S., De Marzio, M., English, B.P., Qi, Y., Jung, S.K., Kieffer-Kwon, K.R., et al. (2020) Nature Methods
To image the accessible genome at nanometer scale in situ, we developed three-dimensional assay for transposase-accessible chromatin-photoactivated localization microscopy (3D ATAC-PALM) that integrates an assay for transposase-accessible chromatin with visualization, PALM super-resolution imaging and lattice light-sheet microscopy. Multiplexed with oligopaint DNA–fluorescence in situ hybridization (FISH), RNA–FISH and protein fluorescence, 3D ATAC-PALM connected microscopy and genomic data, revealing spatially segregated accessible chromatin domains (ACDs) that enclose active chromatin and transcribed genes. Using these methods to analyze genetically perturbed cells, we demonstrated that genome architectural protein CTCF prevents excessive clustering of accessible chromatin and decompacts ACDs. These results highlight 3D ATAC-PALM as a useful tool to probe the structure and organizing mechanism of the genome.
CONDENSIN-DEPENDENT CHROMATIN COMPACTION REPRESSES TRANSCRIPTION GLOBALLY DURING QUIESCENCE
Swygert, S.G., Kim, S., Wu, X., Fu, T., Hsieh, T.H.S., Rando, O.J., Eisenman, R.N., Shendure, J., McKnight, J.N., and Tsukiyama, T. (2018) Molecular Cell
Quiescence is a stress-resistant state in which cells reversibly exit the cell cycle and suspend most processes. Quiescence is essential for stem cell maintenance, and its misregulation is implicated in tumor formation. One of the hallmarks of quiescent cells is highly condensed chromatin. Because condensed chromatin often correlates with transcriptional silencing, it has been hypothesized that chromatin compaction represses transcription during quiescence. However, the technology to test this model by determining chromatin structure within cells at gene resolution has not previously been available. Here, we use Micro-C XL to map chromatin contacts at single-nucleosome resolution genome-wide in quiescent Saccharomyces cerevisiae cells. We describe chromatin domains on the order of 10–60 kilobases that, only in quiescent cells, are formed by condensin-mediated loops. Condensin depletion prevents the compaction of chromatin within domains and leads to widespread transcriptional de-repression. Finally, we demonstrate that condensin-dependent chromatin compaction is conserved in quiescent human fibroblasts.
CHROMATIN DYNAMICS AND THE RNA EXOSOME FUNCTION IN CONCERT TO REGULATE TRANSCRIPTIONAL HOMEOSTASIS
Rege, M., Subramanian, V., Zhu, C., Hsieh, T.H.S., Weiner, A., Friedman, N., Clauder-Münster, S., Steinmetz, L.M., Rando, O.J., Boyer, L.A., et al. (2015) Cell Reports
The histone variant H2A.Z is a hallmark of nucleosomes flanking promoters of protein-coding genes and is often found in nucleosomes that carry lysine 56-acetylated histone H3 (H3-K56Ac), a mark that promotes replication-independent nucleosome turnover. Here, we find that H3-K56Ac promotes RNA polymerase II occupancy at many protein-coding and noncoding loci, yet neither H3-K56Ac nor H2A.Z has a significant impact on steady-state mRNA levels in yeast. Instead, broad effects of H3-K56Ac or H2A.Z on RNA levels are revealed only in the absence of the nuclear RNA exosome. H2A.Z is also necessary for the expression of divergent, promoter-proximal noncoding RNAs (ncRNAs) in mouse embryonic stem cells. Finally, we show that H2A.Z functions with H3-K56Ac to facilitate formation of chromosome interaction domains (CIDs). Our study suggests that H2A.Z and H3-K56Ac work in concert with the RNA exosome to control mRNA and ncRNA expression, perhaps in part by regulating higher-order chromatin structures.
LOSS OF COREPRESSOR PER2 UNDER HYPOXIA UP-REGULATES OCT1-MEDIATED EMT GENE EXPRESSION AND ENHANCES TUMOR MALIGNANCY
Hwang-Verslues, W.W., Chang, P.-H., Jeng, Y.-M., Kuo, W.-H., Chiang, P.-H., Chang, Y.-C., Hsieh, T.H.S., Su, F.-Y., Lin, L.-C., Abbondante, S., et al. (2013) PNAS
The circadian clock gene Period2 (PER2) has been suggested to be a tumor suppressor. However, detailed mechanistic evidence has not been provided to support this hypothesis. We found that loss of PER2 enhanced invasion and activated expression of epithelial-mesenchymal transition (EMT) genes including TWIST1, SLUG, and SNAIL. This finding was corroborated by clinical observation that PER2 down-regulation was associated with poor prognosis in breast cancer patients. We further demonstrated that PER2 served as a transcriptional corepressor, which recruited polycomb proteins EZH2 and SUZ12 as well as HDAC2 to octamer transcription factor 1 (OCT1) (POU2F1) binding sites of the TWIST1 and SLUG promoters to repress expression of these EMT genes. Hypoxia, a condition commonly observed in tumors, caused PER2 degradation and disrupted the PER2 repressor complex, leading to activation of EMT gene expression. This result was further supported by clinical data showing a significant negative correlation between hypoxia and PER2. Thus, our findings clearly demonstrate the tumor suppression function of PER2 and elucidate a pathway by which hypoxia promotes EMT via degradation of PER2.
HDAC2 PROMOTES CELL MIGRATION/INVASION ABILITIES THROUGH HIF‐1Α STABILIZATION IN HUMAN ORAL SQUAMOUS CELL CARCINOMA
Chang, C., Lin, B., Chen, S., Hsieh, T.H.S., Li, Y., and Kuo, M. (2011) Oral Path & Med
Background: Histone deacetylase 2 (HDAC2) expressions in oral squamous cell carcinoma (OSCC) had been implicated in advanced stage and poor prognosis. It suggests a possible link between the migration/invasion potential of oral cancer cells and the prevalent expression of HDAC2.
Methods: Five head and neck cancer (HNC) cell lines, including Ca9‐22, Cal‐27, HSC‐3, SAS, and TW2.6, were used. Cells stably overexpressing HDAC2 and shRNA against HDAC2 were established to investigate migration/invasion ability in vitro and tumorigenesis and progression in vivo.
Results: We found that alterations in the HDAC2 level in OSCC cell lines modulated their invasive ability with a positive correlation. Animal model also showed that knockdown of HDAC2 expression in SAS cells, originally containing high endogenous HDAC2 expression, resulted in decrease in tumor initiation and progression. Using high‐throughput transcriptome analysis, numerous genes involved in HIF‐1α‐associated pathways were found. At the mechanism levels, using agents to block de novo protein synthesis or prevent protein degradation by ubiquitination, we found the stability of hypoxia inducible factor 1α (HIF‐1α) protein was maintained in OSCC cells with HDAC2 overexpression. In addition, co‐immunoprecipitation assay also revealed that HDAC2‐mediated HIF‐1α protein stability is because of direct interaction of HIF‐1α with von Hippel–Lindau (VHL) protein.
Conclusions: Our work demonstrates that HDAC2 maintains HIF‐1α stability, probably at the level of protein modification, which in turn leads to the increase in cell invasion/migration ability in oral cancer progression. These findings implicate the potential of HDAC inhibitors for oral cancer therapy.