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

Embryonic hematopoiesis modulates the inflammatory response and larval hematopoiesis in Drosophila

International audienceRecent lineage tracing analyses have significantly improved our understanding of immune system development and highlighted the importance of the different hematopoietic waves. The current challenge is to understand whether these waves interact and whether this affects the function of the immune system. Here we report a molecular pathway regulating the immune response and involving the communication between embryonic and larval hematopoietic waves in Drosophila. Down-regulating the transcription factor Gcm specific to embryonic hematopoiesis enhances the larval phenotypes induced by over-expressing the pro-inflammatory Jak/Stat pathway or by wasp infestation. Gcm works by modulating the transduction of the Upd cytokines to the site of larval hematopoiesis and hence the response to chronic (Jak/Stat over-expression) and acute (wasp infestation) immune challenges. Thus, homeostatic interactions control the function of the immune system in physiology and pathology. Our data also indicate that a transiently expressed developmental pathway has a long-lasting effect on the immune response

Epigenetic regulation of genes during development: a conserved theme from flies to mammals

Eukaryotic genome is organized in form of chromatin within the nucleus. This organization is important for compaction of DNA as well as for the proper expression of the genes. During early embryonic development, genomic packaging receives variety of signals to eventually set up cell type specific expression patterns of genes. This process of regulated chromatinization leads to "cell type specific epigenomes". The expression states attained during differentiation process need to be maintained subsequently throughout the life of the organism. Epigenetic modifications are responsible for chromatin dependent regulatory mechanism and play a key role in maintenance of the expression state-a process referred to as cellular memory. Another key feature in the packaging of the genome is formation of chromatin domains that are thought to be structural as well as functional units of the higher order chromatin organization. Boundary elements that function to define such domains set the limits of regulatory elements and that of epigenetic modifications. This connection of epigenetic modification, chromatin structure and genome organization has emerged from several studies. Hox genes are among the best studied in this context and have led to the significant understanding of the epigenetic regulation during development. Here we discuss the evolutionarily conserved features of epigenetic mechanisms emerged from studies on homeotic gene clusters

Chromatin remodeling protein INO80 has a role in regulation of homeotic gene expression in Drosophila

The homologues of yeast INO80 are identified across phyla from Caenorhabditis elegans to human. In Drosophila it has been shown that dINO80 forms a complex with Pleiohomeotic but does not interact with Hox PRE (polycomb responsive element). As some proteins of the INO80 complex are implicated in homeotic gene regulation, we examined if dINO80 is involved in regulation of homeotic genes. We find that dINO80 null mutants generated by imprecise excision of P-element are late embryonic lethals and show homeotic transformation. We detect misexpression of homeotic genes like Sex-comb reduced, Antennapedia, Ultrabithorax and Abdominal-B in dIno80 mutant embryos by immunostaining which is further substantiated by quantitative PCR. Polycomb phenotype in dIno80-Pc is enhanced in double mutants. Concurrently, the localization of dINO80 to sequences upstream of misexpressed genes in vivo shows that dINO80 is involved in homeotic gene regulation and probably through its interactions with PcG-trxG complexes

Epigenetic profile of DNMT3L DMC and the associated promoter in the reporter gene assay.

<p>A&B. Histone modifications associated with DNMT3L DMC and the hsp70 promoter in the transgene reporter assay in Drosophila. Histone ChIP analysis for the 3L-L region (A) and the CMV promoter (B) in the Drosophila transgene assay. Chromatin immunoprecipitation was carried out on the 25.2.12 transgenic line with the indicated histone H3 modifications, followed by quantitative Real-time PCR. Comparison of histone modifications associated with the hsp70 promoter in the 3L-L (3LL, black bars) transgenic lines and their flipped out counterparts (Δ3LL, white bars). The H3 histone modifications examined are mentioned below the X-axis. Enrichment in the bound fraction is represented as percentage of Input. IgG - control ChIP with rabbit IgG. Error bars represent Standard Deviation (S.D.). Asterisks indicate significant difference (Student's t test, * - p<0.05, ** - p<0.01, *** - p<0.005).</p

Knock-down of Polycomb proteins and its effect on transcription of the <i>DNMT3L</i> gene from the endogenous locus.

<p>A. siRNA mediated transcriptional repression of polycomb proteins. mRNA levels of the indicated Polycomb genes was assayed in presence of scrambled or specific siRNA in HEK293 cells. % mRNA level was calculated with respect to untransfected cells. B. <i>DNMT3L</i> gene expression from the endogenous locus in Polycomb siRNA transfected or untransfected HEK293 cells was quantitated by Real-time RT-PCR. Relative expression was calculated with respect to untransfected cells. The genes for which siRNA was used in our assay are mentioned below the X-axis. Error bars represent Standard Deviation (S.D.). Asterisks indicate significant difference (Student's t test, * - p<0.05, *** - p<0.001, *** - p<0.005).</p

DNMT3L DMC interacts with Polycomb group of proteins.

<p>A. The 15 panels show comparison of eye color phenotype between representative DNMT3L DMC transgenic lines (25.2.12) and their counterpart lines after crossing with the respective Polyomb (<i>Pc¹</i>, <i>esc²</i>, <i>Asx^XF53</i>, <i>Psc′</i>, <i>Pcl^T1</i>, <i>Scm^R5-13B</i>, <i>Phd</i>, <i>Pho</i>), Trithorax (<i>Ash2¹</i>, <i>Mor¹</i>, <i>Bbrm²</i>, <i>Trl^R85</i>, <i>Trx¹</i>) and Supressor of Variegation (<i>Su(var)2-5⁰¹</i>, <i>Su(var)3-9⁰⁶</i>) mutants. 3LL/+, heterozygous 3L-L transgenic lines; 3LL/−, their respective counterparts after crosses with the respective mutant (− is the name of the Polycomb, Trithorax or Suvar mutant). B. Comparison of eye color pigmentation between 3L-L transgenic lines (25.2.12 and 25.2.29) and their counterparts from crosses with the various mutant lines. Each bar represents eye color pigmentation for progeny from crosses of individual transgenic lines with a particular mutant, the details of which are provided below the X-axis. As the assays were done in batches, the eye pigmentation for the control 3L-L transgenic line was done for each batch and is shown as white bars (P/+ male with <i>W¹¹¹⁸</i>) in the graphs. Error bars represent Standard Deviation (S.D.). Asterisks indicate significant difference (Student's t test, * - p<0.05, ** - p<0.01).</p

Eye color phenotype of the various DNMT3L DMC transgenic lines and their flipped-out counterparts.

<p>The approximate eye color for each line is mentioned. Relative eye pigmentation was calculated as percentage change between each transgenic line and its flipped-out counterpart.</p

Functional analysis of DNMT3L DMC by reporter gene assay in transiently transfected mammalian cells.

<p>A. Graphical representation of the various constructs transfected into HEK293 cells. Shown for each construct is the reporter AcGFP gene, its CMV promoter, the selection Kan^R/Neo^R marker, the various DNMT3L DMC fragments, the control Chr1 fragment and the H19 ICR inserted upstream of the CMV promoter. B. Relative transcriptional level of the reporter AcGFP gene in the various constructs. The transcriptional levels were measured by Real-Time PCR and calculated relative to the AcGFP expression in the control CMV only construct. <i>Kan</i>^R/<i>Neo</i>^R expression level was used to control for transfection efficiency in the Real-Time PCR analysis. C. A representative Western Blot analysis showing AcGFP protein expression for HEK293 cells transfected with the various constructs mentioned in A. β-TUBULIN was used as a loading control. Luciferase activity for the pG5luc vector co-transfected with the above mentioned constructs is shown below the β-TUBULIN panel. D. Relative AcGFP protein expression levels for the cells transfected with the various constructs. Error bars represent Standard Deviation (S.D.). Asterisks indicate significant difference (Student's t test, * - p<0.05, ** - p<0.01, *** - p<0.001).</p

Graphical representation of the alternate Transcription Start Sites for the human <i>DNMT3L</i> gene.

<p>The human <i>DNMT3L</i> gene produces four transcripts (not drawn to scale) from three different Transcription start sites (TSS, raised arrows). Exons are shown as grey rectangles. Shown here in detail are two TSS which are responsible for the DNMT3L-001 & DNMT3L-201 (from +1) and DNMT3L-002 (+334) transcripts. Only DNMT3L-001 and 002 are shown in the representation. The most 5′ TSS has been taken as +1 position. Translation start site (shown as vertical line with ATG) is the same (at +952, in the second exon) for all these three <i>DNMT3L</i> transcripts. However, all the three differ in the length of the final DNMT3L protein. Protein made by DNMT3L-001 and DNMT3L-201 are 387 aa and 386 aa long respectively, DNMT3L-003 is 181 aa and DNMT3L-002 makes a 288 aa long protein. The fragments 3L-L and 3L-S from this region were analysed for their function by reporter gene assays in the present study. The approximate positions of the CpG dinucleotides within these fragments are shown with filled circles. All nucleotide positions mentioned are relative to the most 5′ TSS site.</p