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1 Chromatin and nuclear architecture
Establishment of cell type specific gene expression and epigenetic inheritance of the expression state are essential features of developmental mechanisms. We are studying these processes at the level of higher order chromatin organization. Genomes of higher eukaryotes goes thorough multiple levels of compaction to: a) to fit with in the nuclear space (2 meter human genome fits with 10 micrometer size nucleus), and b) to attain its functional state as this packaging process has regulatory consequences. We are interested in understanding the molecular details of such regulatory mechanisms.
1.1 Molecular analysis of boundary and Polycomb response elements
Enhancers and silencers are known to act across a long distance. Why then do they not misregulate genes in their native context? The most attractive model suggests that genes and their regulatory elements are confined to functionally distinct domains defined by boundary elements. The concept that higher order chromatin organization begins with chromatin domains, the topologically independent structural unit, has stimulated studies aimed to understand how eukaryotic genome is packaged into chromatin and what the functional consequences of this organization are. The idea that boundaries may have an insulating activity has been used to establish in vivo assays for these elements. What is the mechanistic basis of boundary element function? There is no satisfactory answer to this important question as yet. We are analyzing selected boundary elements to dissect the cis motifs and trans acting factors involved in the function of these boundaries in Drosophila.
Epigenetic inheritance to maintain the expression state of the genome is essential during development. In Drosophila, the PREs function to mark the epigenetic cellular memory of the corresponding genomic region with the help of PcG and trxG proteins. While the PcG genes code for the repressor proteins, the trxG genes encode activator proteins. The observations that some proteins may function both as PcG and trxG members and that both these groups of proteins act upon common cis elements indicate at least a partial functional overlap among these proteins. Trl-GAGA was initially identified as a trxG member but later was shown to be essential for PcG function on several PREs. We investigated the mechanism behind the function of Trl-GAGA as a component of the repressive Polycomb system by identifying the direct interacting partners of this protein.
In a yeast two-hybrid screen we identified lola like (lolal), a.k.a. batman, as one of the strong interactors of GAGA factor. lolal also interacts with polyhomeotic (ph) and, like Trl, both lolal and ph are needed for iab-7PRE mediated pairing dependent silencing of a mini-white transgene. Our genetic and biochemical interaction studies suggest that lolal may be the effecter molecule for the Trl-GAGA mediated PcG function.
1.2 The Nuclear matrix
In order to understand structural features of nuclear architecture and to establish a biochemical link between boundary function and the nuclear architecture, we have taken a biochemical approach to analyze the structural components of these elements. To this end, we have initiated biochemical analysis of nuclear matrix that is likely to be the basis for functional compartments and, therefore, key element in the mechanism of the boundary function. We have standardized nuclear matrix preparation protocol with many alterations in the earlier protocols to obtain consistent preparations in a reproducible manner.
NuMat Proteins: We have characterized close to 100 proteins of the nuclear matrix using our proteomics facility. We plan to carry out total proteomics of the nuclear matrix and look at the dynamic aspect of this structure during development. Our data suggests that there is a distinct change in the protein components when 0-2 hour and 14-16 hour embryos are compared. We are analyzing the proteins that show this dynamic appearance.
NuMat RNA: A considerable component of the nuclear matrix is RNA. We have established protocols to isolate such RNAs that are components of nuclear matrix. We are creating a library of these RNAs for further identification and functional studies.
1.3 Genomic organization and chromatin structure in the human Y chromosome
Human Y chromosome offers a convenient system to study genome organization and chromatin structure at chromosomal. The small size, well studied mutations, intermittent blocks of heterochromatin – euchromatin junctions and the complete sequence information are the key advantages with this chromosome.
Bkm (GATA repeats) associated chromatin domain boundaries: The GATA repeats have been shown to be associated with sex chromosomes of various organisms and have been implicated in the evolution and regulatory function of sex chromosomes in snakes and other organisms. Generally, occurrence of simple repeats decreases steadily as the length of repeat increases. In contrast, however, this trend is reversed in case of GATA from six tandem repeat onwards peaking at (GATA) 10-12. Distribution of such longer GATA repeats along the chromosome and their close proximity to Matrix Associated Regions (MARs) suggests that such 'GATA-MAR' regions may be marking chromatin domains for a coordinated expression of genes residing in these domains. We are currently identifying boundary elements from 'GATA-MAR' regions using transgenic 'boundary assays' in Drosophila melanogaster.
Boundaries in the heterochromatin - euchromatin transition zones: The heterochromatin structure begins from the repetitive DNA and it can spread hundreds of kilo bases into nearby unique sequences. In the natural context, therefore, where genes are located within few kilo bases from the repetitive DNA, a boundary must exist between heterochromatin and euchromatin to define a sharp transition zone. Another independent line of observation that strongly argues for the presence of such boundary elements is the presence of genes that reside and get expressed in heterochromatin environment. While it is well known that heterochromatin itself is non-permissive to transcriptional apparatus, it is likely that boundary elements define accessible domains for genes that reside and function in such a context. The q arm of Y chromosome is flanked by heterochromatin structures. Initial analysis on the available sequences from the centromeric heterochromatin and the one distal to Yq, revealed that it mainly consisted of pentameric repeat GGAAT. Using this repeat as landmark, we have mapped the sequences that constitute the transition zone between euchromatin and heterochromatin. We are carrying out extensive analysis of this region to investigate associated sequence features
2 Homeotic gene complexes: The evo-devo of A-P body axis
Homeotic gene complexes determine the anterior-posterior body axis in animals. Expression and function of
the homeotic genes along this axis is colinear with the order of the genes with in the complex. This 'coliniearity' of chromosomal organization and functional correspondence is conserved through out the animal kingdom. Although the molecular basis of this in not understood, it is conceivable that control elements in proximity of these complexes that establish and maintain this colinearity. Several mechanisms have been proposed to link this organization of homeotic genes and the spacio-temporally controlled expression
including the one that involves higher order chromatin organization. Role of chromatin organization in the regulation of homeotic gene complex has been well studied in Drosophila.
2.1 Regulation of bithorax complex of Drosophila melanogaster
Homeotic genes of Drosophila exist in two clusters: the Antennapedia complex (ANT-C) and the bithorax complex (BX-C). Colinearity correspondence of gene order in the complex with the order of body parts that are under the control of these genes, first observed in Drosophila, is conserved up to vertebrates. Vertebrate hox complexes, however, are about ten times more compact as compared to those of insects. BX-C alone spans more than 300 kb, 95% of which constitutes cis regulatory elements. Remarkably though, the colinearity in the BX-C extends even to this vast cis-regulatory region. We have adopted molecular genetic approach to study the regulation of Abd-B region of the BX-C involving higher order chromatin organization. Chromatin domains boundaries and Polycomb response elements are known to be part of this regulatory process. We are interested in understanding the molecular basis of these chromatin elements.
2.2 Evolutionarily conserved features in the organization and regulation of Hox complexes.
The evolution and maintenance of colinear organization of Hox genes is likely to be intimately linked to the mechanism of their regulation and, furthermore, higher chromatin structure may be a key aspect of this mechanism. It is emerging that PRE and "boundary" like elements may be involved in the regulation of mouse HoxD cluster too. We have identified and analyzed such evolutionarily conserved regulatory elements in the HoxD complex.
With the availability of the genomic sequence of several vertebrates, we have carried out annotation of HoxD complex and sequence comparison to identify conserved non-coding regions. This led us to an unprecedented conservation of non-coding DNA sequences adjacent to the HoxD complex of vertebrates. Stretches of hundreds of base pairs in a 7kb region, upstream of HoxD complex, show 100% conservation from fish to human. Using primers designed from these sequences of human HoxD complex, we amplified the corresponding regions from different vertebrates, including mammals, aves, reptiles, amphibians and pisces. Such a high degree of conservation, where no variation was allowed during ~500 million years of evolution, suggests critical function for these sequences in the regulation of the HoxD complex.
3 Comparative genomics of non-coding DNA
In higher eukaryotes a large proportion of the genome does not code for any proteins. Several lines of studies suggest that a large proportion of non-coding DNA may be required for packaging of the genome in the form of chromatin. The regulation of gene expression in higher eukaryotes is very complex and packaging of the genome is suggested to have regulatory consequences. We believe that genomes evolve within this constraint of packaging. We do not know, however, what the 'packaging code' of genome is. Identification and analysis of conserved regulatory elements that work at the level of global regulation of genes through chromatin organization by functioning as chromatin domain boundaries, imprinting/maintenance elements that contribute to cellular memory, etc., are active areas of current research.
3.1 Comparative and functional genomics of non-repetitive non-coding DNA
The newly emerging field of comparative genomics, which is based upon the availability of genome sequences of large number of organisms, opens up a new possibility of identification and analysis of genome sequences. We have started comparative genomics of the non-coding part of genome. Initially, we are comparing selected genetic loci of different species to identify functionally relevant conserved regions. We are also analyzing small chromosomes to understand higher order features of genome organization, packaging and regulatory elements in a 'whole chromosome' context. We have identified unique (non repetitive) regions in these genomes that show extreme conservation but do not resemble any known functional element and hence are likely to be novel class of regulatory elements. We take the genomic context of such sequences as the key indicator of their role in chromatin organization and gene regulation. Following this line of study, we are carrying out computational analysis of genomic sequences to identify patterns within a genome and regions conserved across species.
3.2 Pattern search approach to identify novel regulatory elements
Several chromatin level regulatory elements that have been characterized functionally are being analyzed at molecular level in several laboratories including that of ours. One common theme that emerges from these studies is that there is no significant conservation among these elements although they can often substitute one for the other even across species. What then is the feature that these elements have in common? Several studies suggest that small sequence motifs when present in a cluster may be functionally important. We are developing methods to look for patterns-based clusters of sequence motifs over large stretches of genomic DNA in order to identify such elements.
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