Chromatin Domain Boundaries Research Articles

Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development.

Chromatin domain boundaries delimited by CTCF motifs can restrict the range of enhancer action. However, disruption of domain structure often results in mild gene dysregulation and thus predicting the impact of boundary rearrangements on animal development remains challenging. Here, we tested whether structural perturbation of a chromatin domain with multiple developmental regulators can result in more acute gene dysregulation and severe developmental phenotypes. We targeted clusters of CTCF motifs in a domain of the mouse genome containing three FGF ligand genes - Fgf3, Fgf4, and Fgf15 - that regulate several developmental processes. Deletion of the 23.9kb cluster that defines the centromeric boundary of this domain resulted in ectopic interactions of the FGF genes with enhancers located across the deleted boundary that are active in the developing brain. This caused strong induction of FGF expression and perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary deletion was sufficient to cause these fully penetrant phenotypes, and strikingly, loss of a single CTCF motif within the cluster also recapitulated ectopic FGF expression and caused encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF clusters of this domain, nor to all developmental processes controlled by these three FGF genes - for example, the ability to undergo lineage specification in the blastocyst and pre-implantation development were not affected. By tracing the impact of different chromosomal rearrangements throughout mouse development, we start to uncover the determinants of phenotypic robustness and sensitivity to perturbation of chromatin boundaries. Our data show how small sequence variants at certain domain boundaries can have a surprisingly outsized effect and must be considered as potential sources of gene dysregulation during development and disease.

Abstract 3558: Role of FLI portion of EWS::FLI in transcription regulation via modulation of chromatin 3D landscape in Ewing sarcoma

Abstract Ewing sarcoma is an aggressive bone-associated tumor currently treated with dose-intense chemotherapy, radiation, and surgery and it affects adolescents and young adults. The hallmark of Ewing sarcoma is a translocated fusion transcription factor named EWS::FLI that drives the oncogenic process. We hypothesize that FLI portion of EWS::FLI containing a crucial alpha-helix plays a novel role in transcription regulation of thousands of genes by modulating chromatin looping. The hypothesis is based on recent evidences from our lab: an alpha-helix immediately downstream of DNA binding domain as an important player in regulating transcriptional activity and that EWS::FLI has a substantial role in shaping the chromatin landscape of Ewing sarcoma cells. The objective of this study is to elucidate the mechanism underlying transcriptional regulation by FLI. We utilized knockdown/rescue experiments in which EWS::FLI was depleted with shRNA and replaced with constructs containing the ETS DNA Binding Domain (DBD) alone or DBD+ (DBD and 4th alpha-helix). The binding pattern and transcriptional regulation were assessed with CUT&Tag and RNA-Seq respectively. The extent of roles each of these construct play in organization, structure, and function of chromatin are being assessed using Micro-C technique, a variation of Hi-C with improved resolution, higher signal-to-noise ratio and more information on chromatin domain boundaries and chromatin looping. The DNA binding and genomic localization of EWS::FLI was unaltered by the deletion surrounding the DNA binding domain (which contains a 4th alpha-helix) in A673 and TTC466 cells. Despite this similarity in genomic localization and binding, the transcriptional output driven by EWS::FLI was significantly diminished by the deletion. With this current study, we hope to understand if the flanking region neighboring the DNA binding domain contributes to the chromatin architecture remodeling function of EWS::FLI and whether this function could be attributed to the transcriptional output differences in the DBD and DBD+ conditions. Citation Format: Ariunaa Bayanjargal, Cenny Taslim, Jesse Crow, Julia Selich-Anderson, Stephen L. Lessnick. Role of FLI portion of EWS::FLI in transcription regulation via modulation of chromatin 3D landscape in Ewing sarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3558.

Deciphering Hierarchical Chromatin Domains and Preference of Genomic Position Forming Boundaries in Single Mouse Embryonic Stem Cells.

The exploration of single-cell 3D genome maps reveals that chromatin domains are indeed physical structures presenting in single cells, and domain boundaries vary from cell to cell. However, systematic analysis of the association between regulatory factor binding and elements and the formation of chromatin domains in single cells has not yet emerged. To this end, a hierarchical chromatin domain structure identification algorithm (named as HiCS) is first developed from individual single-cell Hi-C maps, with superior performance in both accuracy and efficiency. The results suggest that in addition to the known CTCF-cohesin complex, Polycomb, TrxG, pluripotent protein families, and other multiple factors also contribute to shaping chromatin domain boundaries in single embryonic stem cells. Different cooperation patterns of these regulatory factors drive genomic position categories with differential preferences forming boundaries, and the most extensive six types of retrotransposons are differentially distributed in these genomic position categories with preferential localization. The above results suggest that these different retrotransposons within genomic regions interplay with regulatory factors navigating the preference of genomic positions forming boundaries, driving the formation of higher-order chromatin structures, and thus regulating cell functions in single mouse embryonic stem cells.

Active enhancers strengthen insulation by RNA-mediated CTCF binding at chromatin domain boundaries.

Vertebrate genomes are partitioned into chromatin domains or topologically associating domains (TADs), which are typically bound by head-to-head pairs of CTCF binding sites. Transcription at domain boundaries correlates with better insulation; however, it is not known whether the boundary transcripts themselves contribute to boundary function. Here we characterize boundary-associated RNAs genome-wide, focusing on the disease-relevant INK4a/ARF and MYC TAD. Using CTCF site deletions and boundary-associated RNA knockdowns, we observe that boundary-associated RNAs facilitate recruitment and clustering of CTCF at TAD borders. The resulting CTCF enrichment enhances TAD insulation, enhancer-promoter interactions, and TAD gene expression. Importantly, knockdown of boundary-associated RNAs results in loss of boundary insulation function. Using enhancer deletions and CRISPRi of promoters, we show that active TAD enhancers, but not promoters, induce boundary-associated RNA transcription, thus defining a novel class of regulatory enhancer RNAs.

Abstract B018: Studying the role of FLI portion of EWS FLI in transcription regulation via modulation of chromatin 3D landscape in Ewing sarcoma

Abstract Ewing sarcoma is an aggressive bone-associated tumor currently treated with dose-intense chemotherapy, radiation, and surgery. Ewing sarcoma affects adolescents and young adults with incidence rate of 3 per million. The hallmark of Ewing sarcoma is a translocated fusion transcription factor named EWS FLI that drives the oncogenic process. The lack of efficient and targeted treatment in Ewing sarcoma directly arises from the poor understanding of how EWS/FLI regulates expression of thousands of genes as well as the current lack of effective targeting of transcription factors. We hypothesize that FLI portion of EWS::FLI containing a crucial alpha-helix plays a novel role in transcription regulation of thousands of genes by modulating chromatin looping. We postulated this hypothesis based on recent evidence from our lab that established an alpha-helix immediately downstream of DNA binding domain as an important player in regulating transcriptional activity in A673 cell line. Structure-function mapping revealed that ETS domain alone was insufficient for full transcriptional regulation. The purpose of this study is to elucidate the mechanism underlying transcriptional regulation by FLI and it is likely to guide future studies on novel therapeutics for Ewing sarcoma patients as transcriptional factors remain elusive targets of therapy. We utilized knockdown/rescue experiments in which EWS FLI was depleted with shRNA and replaced with constructs containing the ETS DNA Binding Domain (DBD) alone or DBD+ (ETS DNA Binding Domain and 4th alpha-helix). The knockdown/rescue experiments were confirmed at mRNA level using qPCR, and at protein level using immunoblots. The binding pattern and transcriptional regulation of DBD and DBD+ were assessed with RNA-Seq respectively. The extent of roles each of these construct play in organization, structure, and function of chromatin are being assessed using Micro-C technique, a variation of Hi-C with improved resolution, higher signal-to-noise ratio and more information on chromatin domain boundaries and chromatin looping. The DNA binding and genomic localization of EWS FLI was unaltered by the deletion surrounding the DNA binding domain (which contains a 4th alpha-helix) in A673 cells. Despite this similarity in genomic localization and binding, the transcriptional output driven by EWS FLI was significantly diminished by the deletion. In addition, we observed this differential transcriptional output by the EWS FLI DBD and DBD+ in another Ewing sarcoma cell line called TTC466. Current work in our lab shows that EWS FLI has a substantial role in shaping the chromatin landscape of Ewing sarcoma cells. We observed this result with Hi-C technique upon the depleting EWS FLI from A673 cells. With this current study, we hope to understand if the flanking region neighboring the DNA binding domain contributes to the chromatin architecture remodeling function of EWS FLI and whether this function could be attributed to the transcriptional output differences in the DBD and DBD+ conditions. Citation Format: Ariunaa Bayanjargal. Studying the role of FLI portion of EWS FLI in transcription regulation via modulation of chromatin 3D landscape in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B018.

The Drosophila BEAF insulator protein interacts with the polybromo subunit of the PBAP chromatin remodeling complex.

The Drosophila Boundary Element-Associated Factor of 32 kDa (BEAF) binds in promoter regions of a few thousand mostly housekeeping genes. BEAF is implicated in both chromatin domain boundary activity and promoter function, although molecular mechanisms remain elusive. Here, we show that BEAF physically interacts with the polybromo subunit (Pbro) of PBAP, a SWI/SNF-class chromatin remodeling complex. BEAF also shows genetic interactions with Pbro and other PBAP subunits. We examine the effect of this interaction on gene expression and chromatin structure using precision run-on sequencing and micrococcal nuclease sequencing after RNAi-mediated knockdown in cultured S2 cells. Our results are consistent with the interaction playing a subtle role in gene activation. Fewer than 5% of BEAF-associated genes were significantly affected after BEAF knockdown. Most were downregulated, accompanied by fill-in of the promoter nucleosome-depleted region and a slight upstream shift of the +1 nucleosome. Pbro knockdown caused downregulation of several hundred genes and showed a correlation with BEAF knockdown but a better correlation with promoter-proximal GAGA factor binding. Micrococcal nuclease sequencing supports that BEAF binds near housekeeping gene promoters while Pbro is more important at regulated genes. Yet there is a similar general but slight reduction of promoter-proximal pausing by RNA polymerase II and increase in nucleosome-depleted region nucleosome occupancy after knockdown of either protein. We discuss the possibility of redundant factors keeping BEAF-associated promoters active and masking the role of interactions between BEAF and the Pbro subunit of PBAP in S2 cells. We identify Facilitates Chromatin Transcription (FACT) and Nucleosome Remodeling Factor (NURF) as candidate redundant factors.

Deciphering the regulatory logic of a chromatin domain boundary.

Deciphering the regulatory logic of a chromatin domain boundary.

Detection of Allele-Specific 3D Chromatin Interactions Using High-Resolution In-Nucleus 4C-seq.

Chromosome conformation capture techniques are a set of methods used to determine 3D genome organization through the capture and identification of physical contacts between pairs of genomic loci. Among them, 4C-seq (circular chromosome conformation capture coupled to high-throughput sequencing) allows for the identification and quantification of the sequences interacting with a preselected locus of interest. 4C-seq has been widely used in the literature, mainly to study chromatin loops between enhancers and promoters or between CTCF binding sites and to identify chromatin domain boundaries. As 3D-contacts may be established in an allele-specific manner, we describe an up-to-date allele-specific 4C-seq protocol, starting from the selection of allele-specific viewpoints to Illumina sequencing. This protocol has mainly been optimized for cultured mammalian cells, but can be adapted for other cell types with relatively minor changes in initial steps.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

CTCF and transcription influence chromatin structure re-configuration after mitosis

During mitosis, transcription is globally attenuated and chromatin architecture is dramatically reconfigured. We exploited the M- to G1-phase progression to interrogate the contributions of the architectural factor CTCF and the process of transcription to genome re-sculpting in newborn nuclei. Depletion of CTCF during the M- to G1-phase transition alters short-range compartmentalization after mitosis. Chromatin domain boundary re-formation is impaired upon CTCF loss, but a subset of boundaries, characterized by transitions in chromatin states, is established normally. Without CTCF, structural loops fail to form, leading to illegitimate contacts between cis-regulatory elements (CREs). Transient CRE contacts that are normally resolved after telophase persist deeply into G1-phase in CTCF-depleted cells. CTCF loss-associated gains in transcription are often linked to increased, normally illegitimate enhancer-promoter contacts. In contrast, at genes whose expression declines upon CTCF loss, CTCF seems to function as a conventional transcription activator, independent of its architectural role. CTCF-anchored structural loops facilitate formation of CRE loops nested within them, especially those involving weak CREs. Transcription inhibition does not significantly affect global architecture or transcription start site-associated boundaries. However, ongoing transcription contributes considerably to the formation of gene domains, regions of enriched contacts along gene bodies. Notably, gene domains emerge in ana/telophase prior to completion of the first round of transcription, suggesting that epigenetic features in gene bodies contribute to genome reconfiguration prior to transcription. The focus on the de novo formation of nuclear architecture during G1 entry yields insights into the contributions of CTCF and transcription to chromatin architecture dynamics during the mitosis to G1-phase progression.

Combinatorial CRISPR inversions of CTCF sites in HOXD cluster reveal complex insulator function

CTCF (CCCTC-binding factor) is a zinc-finger protein which plays a vital role in the three-dimensional (3D) genome architecture. A pair of forward-reverse convergent CTCF binding sites (CBS elements) mediates long-distance DNA interactions to form chromatin loops with the assistance of the cohesin complex, while CBS elements at the chromatin domain boundaries show reverse-forward divergent patterns and function as insulators to discriminate against DNA interaction between chromatin domains. However, there are still many unresolved problems regarding CTCF-mediated insulation function. In order to study the connections between chromatin loops and the insulation function of CBS elements, we combinatorically inverted CBS elements at the HOXD locus by using CRISPR/Cas9 DNA-fragment editing methods in the HEK293T cell line and obtained five different kinds of single-cell CRISPR clones. By performing quantitative high-resolution chromosome conformation capture copy (QHR-4C) experiments, we found that boundary CBS inversions abolish original chromatin loops and establish new loops from the opposite direction, thus shifting the insulator boundary to the new divergent CTCF sites. Furthermore, tandem CBS elements block cohesin permeated from the opposite orientation to function as insulators. RNA-seq experiments showed that alterations of local three-dimensional genome architecture would further influence gene expression of the HOXD cluster. In conclusion, a pair of divergent CBS elements function as insulators by forming chromatin loops within chromatin domains to block cohesin sliding.

Enhancer release and retargeting activates disease-susceptibility genes.

The functional engagement between an enhancer and its target promoter ensures precise gene transcription1. Understanding the basis of promoter choice by enhancers has important implications for health and disease. Here we report that functional loss of a preferred promoter can release its partner enhancer to loop to and activate an alternative promoter (or alternative promoters) in the neighbourhood. We refer to this target-switching process as 'enhancer release and retargeting'. Genetic deletion, motif perturbation or mutation, and dCas9-mediated CTCF tethering reveal that promoter choice by an enhancer can be determined by the binding of CTCF at promoters, in a cohesin-dependent manner-consistent with a model of 'enhancer scanning' inside the contact domain. Promoter-associated CTCF shows a lower affinity than that at chromatin domain boundaries and often lacks a preferred motif orientation or a partnering CTCF at the cognate enhancer, suggesting properties distinct from boundary CTCF. Analyses of cancer mutations, data from the GTEx project and risk loci from genome-wide association studies, together with a focused CRISPR interference screen, reveal that enhancer release and retargeting represents an overlooked mechanism that underlies the activation of disease-susceptibility genes, as exemplified by a risk locus for Parkinson's disease (NUCKS1-RAB7L1) and three loci associated with cancer (CLPTM1L-TERT, ZCCHC7-PAX5 and PVT1-MYC).

CTCF mediates dosage- and sequence-context-dependent transcriptional insulation by forming local chromatin domains.

Insulators play a critical role in spatiotemporal gene regulation in animals. The evolutionarily conserved CCCTC-binding factor (CTCF) is required for insulator function in mammals, but not all of its binding sites act as insulators. Here we explore the sequence requirements of CTCF-mediated transcriptional insulation using a sensitive insulator reporter in mouse embryonic stem cells. We find that insulation potency depends on the number of CTCF-binding sites in tandem. Furthermore, CTCF-mediated insulation is dependent on upstream flanking sequences at its binding sites. CTCF-binding sites at topologically associating domain boundaries are more likely to function as insulators than those outside topologically associating domain boundaries, independently of binding strength. We demonstrate that insulators form local chromatin domain boundaries and weaken enhancer-promoter contacts. Taken together, our results provide genetic, molecular and structural evidence connecting chromatin topology to the action of insulators in the mammalian genome.

Independence of chromatin conformation and gene regulation during Drosophila dorsoventral patterning

The relationship between chromatin organization and gene regulation remains unclear. While disruption of chromatin domains and domain boundaries can lead to misexpression of developmental genes, acute depletion of regulators of genome organization has a relatively small effect on gene expression. It is therefore uncertain whether gene expression and chromatin state drive chromatin organization or whether changes in chromatin organization facilitate cell-type-specific activation of gene expression. Here, using the dorsoventral patterning of the Drosophila melanogaster embryo as a model system, we provide evidence for the independence of chromatin organization and dorsoventral gene expression. We define tissue-specific enhancers and link them to expression patterns using single-cell RNA-seq. Surprisingly, despite tissue-specific chromatin states and gene expression, chromatin organization is largely maintained across tissues. Our results indicate that tissue-specific chromatin conformation is not necessary for tissue-specific gene expression but rather acts as a scaffold facilitating gene expression when enhancers become active.

Genome-Wide Histone Modifications and CTCF Enrichment Predict Gene Expression in Sheep Macrophages.

Alveolar macrophages function in innate and adaptive immunity, wound healing, and homeostasis in the lungs dependent on tissue-specific gene expression under epigenetic regulation. The functional diversity of tissue resident macrophages, despite their common myeloid lineage, highlights the need to study tissue-specific regulatory elements that control gene expression. Increasing evidence supports the hypothesis that subtle genetic changes alter sheep macrophage response to important production pathogens and zoonoses, for example, viruses like small ruminant lentiviruses and bacteria like Coxiella burnetii. Annotation of transcriptional regulatory elements will aid researchers in identifying genetic mutations of immunological consequence. Here we report the first genome-wide survey of regulatory elements in any sheep immune cell, utilizing alveolar macrophages. We assayed histone modifications and CTCF enrichment by chromatin immunoprecipitation with deep sequencing (ChIP-seq) in two sheep to determine cis-regulatory DNA elements and chromatin domain boundaries that control immunity-related gene expression. Histone modifications included H3K4me3 (denoting active promoters), H3K27ac (active enhancers), H3K4me1 (primed and distal enhancers), and H3K27me3 (broad silencers). In total, we identified 248,674 reproducible regulatory elements, which allowed assignment of putative biological function in macrophages to 12% of the sheep genome. Data exceeded the FAANG and ENCODE standards of 20 million and 45 million useable fragments for narrow and broad marks, respectively. Active elements showed consensus with RNA-seq data and were predictive of gene expression in alveolar macrophages from the publicly available Sheep Gene Expression Atlas. Silencer elements were not enriched for expressed genes, but rather for repressed developmental genes. CTCF enrichment enabled identification of 11,000 chromatin domains with mean size of 258 kb. To our knowledge, this is the first report to use immunoprecipitated CTCF to determine putative topological domains in sheep immune cells. Furthermore, these data will empower phenotype-associated mutation discovery since most causal variants are within regulatory elements.

A pitfall for machine learning methods aiming to predict across cell types

Machine learning models that predict genomic activity are most useful when they make accurate predictions across cell types. Here, we show that when the training and test sets contain the same genomic loci, the resulting model may falsely appear to perform well by effectively memorizing the average activity associated with each locus across the training cell types. We demonstrate this phenomenon in the context of predicting gene expression and chromatin domain boundaries, and we suggest methods to diagnose and avoid the pitfall. We anticipate that, as more data becomes available, future projects will increasingly risk suffering from this issue.

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

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.

Identification of cis Elements for Spatio-temporal Control of DNA Replication

The temporal order of DNA replication (replication timing, RT) is highly coupled with genome architecture, but cis-elements regulating spatio-temporal control of replicatio have remained elusive. We performed an extensive series of CRISPR mediated deletions and inversions and high-resolution capture Hi-C of a pluripotency associated domain (DppA2/4) in mouse embryonic stem cells. Whereas CTCF mediated loops and chromatin domain boundaries were dispensable, deletion of three intra-domain prominent CTCF-independent 3D contact sites caused a domain-wide delay in RT, shift in sub-nuclear chromatin compartment and loss of transcriptional activity, These “early replication control elements” (ERCEs) display prominent chromatin features resembling enhancers/promoters and individual and pair-wise deletions of the ERCEs confirmed their partial redundancy and interdependency in controlling domain-wide RT and transcription. Our results demonstrate that discrete cis-regulatory elements mediate domain-wide RT, chromatin compartmentalization, and transcription, representing a major advance in dissecting the relationship between genome structure and function.

Genomic organization of Polycomb Response Elements and its functional implication in Drosophila and other insects

The epigenetic memory is an essential aspect of multicellular organisms to maintain several cell types and their gene expression pattern. This complex process uses a number of protein factors and specific DNA elements within the developmental cues to achieve this. The protein factors involved in the process are the Polycomb group (PcG) members, and, accordingly, the DNA sequences that interact with these proteins are called Polycomb Response Elements (PREs). Since the PcG proteins are highly conserved among higher eukaryotes, including insects, and function at thousands of sites in the genomes, it is expected that PREs mayalso be present across the genome.However, the studies on PREs in insect species, other thanDrosophila, is currently lacking.We took a bioinformatics approach to develop an inclusive PRE prediction tool, 'PRE Mapper', to address this need. By applying this tool on the Drosophila melanogaster genome, we predicted greater than 20,000 PREs.When comparedwith the available PRE prediction methods, this tool shows far better performance by correctly identifying the in vivo binding sites of PcG proteins, identified by genome-scale ChIP experiments. Further analysis of the predicted PREs shows their cohabitation with chromatin domain boundary elements at several places in the Drosophila genome, possibly defining a composite epigenetic module.We analysed 10 insect genomes in this context and find several conserved features in PREs across the insect species with some variations in their occurrence frequency. These analyses leading to the identification of PREin insect genomes contribute to our understanding of epigenetic mechanisms in these organisms.

Lessons on gene regulation learnt from the Drosophila melanogaster bithorax complex.

Homeotic or Hox genes determine the anterior-posterior body axis in all bilaterians. As expected, Hox genes are highly conserved across bilaterians. Interestingly, however, the peculiar organization of Hox genes in the form of clusters where the order of occurrence of genes in the genome corresponds to the order in which they regulate segmental identity of anterior-posterior body axis is also conserved. The relation between collinearity of arrangement of genes on the chromosomes and spatial function along the body axis has attracted attention to exploring its relevance in the precise regulation of Hox genes. Conservation of genes and their arrangement suggests a linkage between co-regulation and the higher order chromatin organization of the entire complex. To this end, we and others have used Drosophila as the model system to understand the cis-and trans-regulatory components of Hox genes. A number of chromatin-level regulatory elements, like chromatin domain boundaries, and Polycomb Response Elements (PREs) have been discovered in this process. Interestingly, much of what has emerged from the study of homeotic genes, the cis-elements and protein factors, have relevance across the genome in a large number of regulatory events beyond the Hox genes. Here, we review our findings and discuss their genome-wide implications in complex regulatory processes.