An Organizational Hub of Developmentally Regulated Chromatin Loops in the Drosophila Antennapedia Complex

Chromatin boundary elements (CBEs) are widely distributed in the genome and mediate formation of chromatin loops, but their roles in gene regulation remain poorly understood. The complex expression pattern of the Drosophila homeotic gene Sex combs reduced (Scr) is directed by an unusually long regulatory sequence harboring diverse cis elements and an intervening neighbor gene fushi tarazu (ftz). Here we report the presence of a multitude of CBEs in the Scr regulatory region. Selective and dynamic pairing among these CBEs mediates developmentally regulated chromatin loops. In particular, the SF1 boundary plays a central role in organizing two subsets of chromatin loops: one subset encloses ftz, limiting its access by the surrounding Scr enhancers and compartmentalizing distinct histone modifications, and the other subset subdivides the Scr regulatory sequences into independent enhancer access domains. We show that these CBEs exhibit diverse enhancer-blocking activities that vary in strength and tissue distribution. Tandem pairing of SF1 and SF2, two strong CBEs that flank the ftz domain, allows the distal enhancers to bypass their block in transgenic Drosophila, providing a mechanism for the endogenous Scr enhancer to circumvent the ftz domain. Our study demonstrates how an endogenous CBE network, centrally orchestrated by SF1, could remodel the genomic environment to facilitate gene regulation during development.     

Tracking and interpreting long-range chromatin interactions with super-resolution live-cell imaging

Mammalian genomes are organized and regulated through long-range chromatin interactions. Structural loops formed by CCCTC-binding factor (CTCF) and cohesin fold the genome into domains, while enhancers interact with promoters across vast genomic distances to regulate gene expression. Although genomics and fixed-cell imaging approaches help illuminate many aspects of chromatin interactions, temporal information is usually lost. Here, we discuss how 3D super-resolution live-cell imaging (SRLCI) can resolve open questions on the dynamic formation and dissolution of chromatin interactions. We discuss SRLCI experimental design, implementation strategies, and data interpretation and highlight associated pitfalls. We conclude that, while technically demanding, SRLCI approaches will likely emerge as a critical tool to dynamically probe 3D genome structure and function and to study enhancer–promoter interactions and chromatin looping.     

障碍子机制研究进展

在真核细胞 ,尽管一些转录活性基因和转录沉默基因的调控序列位置常常非常接近 ,它们的表达却并不互相影响。这是由于一些边界元件将染色体分成了不同的区域。障碍子就是边界元件的一种 ,它可以使活性基因的表达免受异染色质的作用 ,防止基因沉默。概述障碍子的作用机制和作用模型。     

Chromatin loops in gene regulation

The control of gene expression involves regulatory elements that can be very far from the genes they control. Several recent technological advances have allowed the direct detection of chromatin loops that juxtapose distant genomic sites in the nucleus. Here we review recent studies from various model organisms that have provided new insights into the functions of chromatin loops and the mechanisms that form them. We discuss the widespread impact of chromatin loops on gene activation, repression, genomic imprinting and the function of enhancers and insulators.     

Crosstalk between histone modifications maintains the developmental pattern of gene expression on a tissue-specific locus

Genome wide studies have provided a wealth of information related to histone modifications. Particular modifications, which can encompass both broad and discrete regions, are associated with certain genomic elements and gene expression status. Here we focus on how studies on the ß-globin gene cluster can complement the genome wide effort through the thorough dissection of histone modifying protein crosstalk. The ß-globin locus serves as a model system to study both regulation of gene expression driven at a distance by enhancers and mechanisms of developmental switching of clustered genes. We investigate recent studies, which uncover that histone methyltransferases, recruited at the ß-globin enhancer, control gene expression by long range propagation on chromatin. Specifically, we focus on how seemingly antagonistic complexes, such as those including MLL2, G9a and UTX, can cooperate to functionally regulate developmentally controlled gene expression. Finally, we speculate on the mechanisms of chromatin modifying complex propagation on genomic domains.     

Conserved boundary elements from the Hox complex of mosquito,Anopheles gambiae

The conservation of hox genes as well as their genomic organization across the phyla suggests that this system of anterior–posterior axis formation arose early during evolution and has come under strong selection pressure. Studies in the split Hox cluster of Drosophila have shown that proper expression of hox genes is dependent on chromatin domain boundaries that prevent inappropriate interactions among different types of cis-regulatory elements. To investigate whether boundary function and their role in regulation of hox genes is conserved in insects with intact Hox clusters, we used an algorithm to locate potential boundary elements in the Hox complex of mosquito, Anopheles gambiae. Several potential boundary elements were identified that could be tested for their functional conservation. Comparative analysis revealed that like Drosophila, the bithorax region in A. gambiae contains an extensive array of boundaries and enhancers organized into domains. We analysed a subset of candidate boundary elements and show that they function as enhancer blockers in Drosophila. The functional conservation of boundary elements from mosquito in fly suggests that regulation of hox genes involving chromatin domain boundaries is an evolutionary conserved mechanism and points to an important role of such elements in key developmentally regulated loci.     

Global chromatin domain organization of the Drosophila genome

In eukaryotes, neighboring genes can be packaged together in specific chromatin structures that ensure their coordinated expression. Examples of such multi-gene chromatin domains are well-documented, but a global view of the chromatin organization of eukaryotic genomes is lacking. To systematically identify multi-gene chromatin domains, we constructed a compendium of genome-scale binding maps for a broad panel of chromatin-associated proteins in Drosophila melanogaster. Next, we computationally analyzed this compendium for evidence of multi-gene chromatin domains using a novel statistical segmentation algorithm. We find that at least 50% of all fly genes are organized into chromatin domains, which often consist of dozens of genes. The domains are characterized by various known and novel combinations of chromatin proteins. The genes in many of the domains are coregulated during development and tend to have similar biological functions. Furthermore, during evolution fewer chromosomal rearrangements occur inside chromatin domains than outside domains. Our results indicate that a substantial portion of the Drosophila genome is packaged into functionally coherent, multi-gene chromatin domains. This has broad mechanistic implications for gene regulation and genome evolution.     

Evidences for insulator activity of the 5′UTR of the Drosophila melanogaster LTR-retrotransposon ZAM

Insulators or chromatin boundary are DNA elements that organize the genome into discrete regulatory domains by limiting the actions of enhancers and silencers through a “positional-blocking mechanism”. The role of these sequences, both in modulation of the enhancers range of action (enhancer–promoter selectivity) and in the organization of the chromatin in functional domains, is emerging strongly in these last years. There is a great interest in identifying new insulators because deeper knowledge of these elements can help understand how cis-regulatory elements coordinate the expression of the target genes. However, while insulators are critical in gene regulation and genome functioning, only a few have been reported so far. Here, we describe a new insulator sequence that is located in the 5′UTR of the Drosophila retrotransposon ZAM. We have used an “enhancer–blocking assay” to test its effects on the activity of the enhancer in transiently transfected Drosophila S2R⁺ cell line. Moreover, we show that the new insulator is able to affect significantly the enhancer–promoter interaction in the human cell line HEK293. These results suggest the possibility of employing the ZAM insulator in gene transfer protocols from insects to mammals in order to counteract the transgene positional and genotoxic effects.     

Relaxed 3D genome conformation facilitates the pluripotent to totipotent-like state transition in embryonic stem cells

The 3D genome organization is crucial for gene regulation. Although recent studies have revealed a uniquely relaxed genome conformation in totipotent early blastomeres of both fertilized and cloned embryos, how weakened higher-order chromatin structure is functionally linked to totipotency acquisition remains elusive. Using low-input Hi-C, ATAC-seq and ChIP-seq, we systematically examined the dynamics of 3D genome and epigenome during pluripotent to totipotent-like state transition in mouse embryonic stem cells (ESCs). The spontaneously converted 2-cell-embryo-like cells (2CLCs) exhibited more relaxed chromatin architecture compared to ESCs, including global weakening of both enhancer-promoter interactions and TAD insulation. While the former correlated with inactivation of ESC enhancers and down-regulation of pluripotent genes, the latter might facilitate contacts between the putative new enhancers arising in 2CLCs and neighboring 2C genes. Importantly, disruption of chromatin organization by depleting CTCF or the cohesin complex promoted the ESC to 2CLC transition. Our results thus establish a critical role of 3D genome organization in totipotency acquisition.