Surabhi Srivastava

Vertebrate GAGA factor associated insulator elements demarcate homeotic genes in the HOX clusters

BackgroundHox genes impart segment identity to body structures along the anterior-posterior axis and are crucial for the proper development of all organisms. Multiple regulatory elements, best defined in Drosophila melanogaster, ensure that Hox expression patterns follow the spatial and temporal colinearity reflected in their tight genomic organization. However, the precise mechanisms that regulate colinear patterns of Hox gene expression remain unclear, especially in higher vertebrates where it is not fully determined how the distinct activation domains of the tightly clustered Hox genes are defined independently of each other. Here, we report the identification of a large number of novel cis-elements at mammalian Hox clusters that can help in regulating their precise expression pattern.ResultsWe have identified DNA elements at all four murine Hox clusters that show poor association with histone H3 in chromatin immunoprecipitation (ChIP)-chip tiling arrays. The majority of these elements lie in the intergenic regions segregating adjacent Hox genes; we demonstrate that they possess efficient enhancer-blocking activity in mammalian cells. Further, we find that these histone-free intergenic regions bear GA repeat motifs and associate with the vertebrate homolog of the GAGA binding boundary factor. This suggests that they can act as GAGA factor-dependent chromatin boundaries that create independent domains, insulating each Hox gene from the influence of neighboring regulatory elements.ConclusionsOur results reveal a large number of potential regulatory elements throughout the murine Hox clusters. We further demarcate the precise location of several novel cis-elements bearing chromatin boundary activity that appear to segregate successive Hox genes. This reflects a pattern reminiscent of the organization of homeotic genes in Drosophila, where such regulatory elements have been characterized. Our findings thus provide new insights into the regulatory processes and evolutionarily conserved epigenetic mechanisms that control homeotic gene expression.

Regulation of cellular chromatin state: insights from quiescence and differentiation.

The identity and functionality of eukaryotic cells is defined not just by their genomic sequence which remains constant between cell types, but by their gene expression profiles governed by epigenetic mechanisms. Epigenetic controls maintain and change the chromatin state throughout development, as exemplified by the setting up of cellular memory for the regulation and maintenance of homeotic genes in proliferating progenitors during embryonic development. Higher order chromatin structure in reversibly arrested adult stem cells also involves epigenetic regulation and in this review we highlight common trends governing chromatin states, focusing on quiescence and differentiation during myogenesis. Together, these diverse developmental modules reveal the dynamic nature of chromatin regulation providing fresh insights into the role of epigenetic mechanisms in potentiating development and differentiation.

A functionally conserved boundary element from the mouse HoxD locus requires GAGA factor in Drosophila

Hox genes are necessary for proper morphogenesis and organization of various body structures along the anterior-posterior body axis. These genes exist in clusters and their expression pattern follows spatial and temporal co-linearity with respect to their genomic organization. This colinearity is conserved during evolution and is thought to be constrained by the regulatory mechanisms that involve higher order chromatin structure. Earlier studies, primarily in Drosophila, have illustrated the role of chromatin-mediated regulatory processes, which include chromatin domain boundaries that separate the domains of distinct regulatory features. In the mouse HoxD complex, Evx2 and Hoxd13 are located ∼9 kb apart but have clearly distinguishable temporal and spatial expression patterns. Here, we report the characterization of a chromatin domain boundary element from the Evx2-Hoxd13 region that functions in Drosophila as well as in mammalian cells. We show that the Evx2-Hoxd13 region has sequences conserved across vertebrate species including a GA repeat motif and that the Evx2-Hoxd13 boundary activity in Drosophila is dependent on GAGA factor that binds to the GA repeat motif. These results show that Hox genes are regulated by chromatin mediated mechanisms and highlight the early origin and functional conservation of such chromatin elements.

Epigenetic profile of the euchromatic region of human Y chromosome

The genome of a multi-cellular organism acquires various functional capabilities in different cell types by means of distinct chromatin modifications and packaging states. Acquired during early development, the cell type-specific epigenotype is maintained by cellular memory mechanisms that involve epigenetic modifications. Here we present the epigenetic status of the euchromatic region of the human Y chromosome that has mostly been ignored in earlier whole genome epigenetic mapping studies. Using ChIP-on-chip approach, we mapped H3K9ac, H3K9me3, H3K27me3 modifications and CTCF binding sites while DNA methylation analysis of selected CpG islands was done using bisulfite sequencing. The global pattern of histone modifications observed on the Y chromosome reflects the functional state and evolutionary history of the sequences that constitute it. The combination of histone and DNA modifications, along with CTCF association in some cases, reveals the transcriptional potential of all protein coding genes including the sex-determining gene SRY and the oncogene TSPY. We also observe preferential association of histone marks with different tandem repeats, suggesting their importance in genome organization and gene regulation. Our results present the first large scale epigenetic analysis of the human Y chromosome and link a number of cis-elements to epigenetic regulatory mechanisms, enabling an understanding of such mechanisms in Y chromosome linked disorders.

A ChIP-on-chip tiling array approach detects functional histone-free regions associated with boundaries at vertebrate HOX genes.

Hox genes impart segment identity to body structures along the anterior–posterior axis and are crucial for proper development. A unique feature of the Hox loci is the collinearity between the gene position within the cluster and its spatial expression pattern along the body axis. However, the mechanisms that regulate collinear patterns of Hox gene expression remain unclear, especially in higher vertebrates. We recently identified novel histone-free regions (HFRs) that can act as chromatin boundary elements demarcating successive murine Hox genes and help regulate their precise expression domains (Srivastava et al., 2013). In this report, we describe in detail the ChIP-chip analysis strategy associated with the identification of these HFRs. We also provide the Perl scripts for HFR extraction and quality control analysis for this custom designed tiling array dataset.

C-State: an interactive web app for simultaneous multi-gene visualization and comparative epigenetic pattern search

BackgroundComparative epigenomic analysis across multiple genes presents a bottleneck for bench biologists working with NGS data. Despite the development of standardized peak analysis algorithms, the identification of novel epigenetic patterns and their visualization across gene subsets remains a challenge.ResultsWe developed a fast and interactive web app, C-State (Chromatin-State), to query and plot chromatin landscapes across multiple loci and cell types. C-State has an interactive, JavaScript-based graphical user interface and runs locally in modern web browsers that are pre-installed on all computers, thus eliminating the need for cumbersome data transfer, pre-processing and prior programming knowledge.ConclusionsC-State is unique in its ability to extract and analyze multi-gene epigenetic information. It allows for powerful GUI-based pattern searching and visualization. We include a case study to demonstrate its potential for identifying user-defined epigenetic trends in context of gene expression profiles.

Epigenetic factors Polycomb ( Pc ) and Suppressor of zeste ( Su ( z )2) negatively regulate longevity in Drosophila melanogaster

The process of aging is a hallmark of the natural life span of all organisms and individuals within a population show variability in the measures of age related performance. Longevity and the rate of aging are influenced by several factors such as genetics, nutrition, stress, and environment. Many studies have focused on the genes that impact aging and there is increasing evidence that epigenetic factors regulate these genes to control life span. Polycomb (PcG) and trithorax (trxG) protein complexes maintain the expression profiles of developmentally important genes and regulate many cellular processes. Here, we report that mutations of PcG and trxG members affect the process of aging in Drosophila melanogaster, with perturbations mostly associated with retardation in aging. We find that mutations in polycomb repressive complex (PRC1) components Pc and Su(z)2 increase fly survival. Using an inducible UAS-GAL4 system, we show that this effect is tissue-specific; knockdown in fat body, but not in muscle or brain tissues, enhances life span. We hypothesize that these two proteins influence life span via pathways independent of their PRC1 functions, with distinct effects on response to oxidative stress. Our observations highlight the role of global epigenetic regulators in determining life span.

PDGFRα signaling in cardiac stem and stromal cells modulates quiescence, metabolism and self-renewal, and promotes anatomical and functional repair

The interstitial and perivascular spaces of the mammalian heart contain a highly interactive tissue community essential for cardiac homeostasis, repair and regeneration. Mesenchymal cells (fibroblasts) are one of the most abundant cell types and make up the organ superstructure. They play key roles as sentinels, tissue architects, paracrine signaling hubs and lineage precursors, and are linked to heart disease through their roles in inflammation and fibrosis. Platelet-derived growth factors (PFGFs) are secreted by several cell types involved in cardiac injury and repair. Here we explore in detail how PDGF receptor α (PDGFRα) signaling in cardiac fibroblasts affects their activation, differentiation and self-renewal. Short-term systemic delivery of PDGF-AB to mice from the time of myocardial infarction (MI) led to activation, proliferation and increased self-renewal of fibroblasts, and subsequent expansion of myofibroblasts. Underpinning this, as evidenced in uninjured mice, was a cell cycle priming effect, in which PDGF-AB induced exit of fibroblasts from quiescence without stimulating proliferation. Despite fibrogenesis, however, treated MI hearts showed enhanced anatomical and functional repair that was associated with pro-regenerative effects on endothelial cells, immune cells and cardiomyocytes. Our study provides a rationale for a novel therapeutic approach to cardiac injury involving stimulating fibroblast activation and endogenous repair mechanisms.

The placental gateway of maternal transgenerational epigenetic inheritance

While much of our understanding of genetic inheritance is based on the genome of the organism, it is becoming clear that there is an ample amount of epigenetic inheritance, which though reversible, escapes erasing process during gametogenesis and goes on to the next generation. Several examples of transgenerational inheritance of epigenetic features with potential impact on embryonic development and subsequent adult life have come to light. In placental mammals, the placenta is an additional route for epigenetic information flow. This information does not go through any meiotic reprogramming and is, therefore, likely to have a more profound influence on the organism. This also has the implication of providing epigenetic instructions for several months, which is clearly a maternal advantage. Although less well-known, there is also an impact of the embryo in emitting genetic information to the maternal system that remains well beyond the completion of the pregnancy. In this review, we discuss several factors in the context of the evolution of this mammal-specific phenomenon, including genomic imprinting, micromosaicism, and assisted reproduction. We also highlight how this kind of inheritance might require attention in the modern lifestyle within the larger context of the evolutionary process.