Project description:Genome organization is essential for proper function, including gene expression. In metazoan genome organization, chromatin loops and Topologically Associated Domains (TADs) facilitate local gene clustering, while chromosomes form distinct nuclear territories characterized by compartmentalization of silent heterochromatin at the nuclear periphery and active euchromatin in the nucleus center. A similar hierarchical organization occurs in the fungus Neurospora crassa where its seven chromosomes form a Rabl conformation, where heterochromatic centromeres and telomeres independently cluster at the nuclear membrane, while interspersed heterochromatic loci in Neurospora aggregate across megabases of linear genomic distance for forming TAD-like structures. However, the role of individual heterochromatic loci in normal genome organization and function is unknown. Here, we examined the genome organization of a Neurospora strain harboring a ~41 kilobase facultative (temporarily silent) heterochromatic region deletion, as well as the genome organization of a strain deleted of a ~110 kilobase permanently silent constitutive heterochromatic region. While the facultative heterochromatin deletion had little effect on local chromatin structure, the constitutive heterochromatin deletion altered local TAD-like structures, gene expression, and the predicted 3D genome structure by qualitatively repositioning genes into the nucleus center. Our work elucidates the role of individual heterochromatic regions for genome organization and function.
Project description:Genome organization is essential for proper function, including gene expression. In metazoan genome organization, chromatin loops and Topologically Associated Domains (TADs) facilitate local gene clustering, while chromosomes form distinct nuclear territories characterized by compartmentalization of silent heterochromatin at the nuclear periphery and active euchromatin in the nucleus center. A similar hierarchical organization occurs in the fungus Neurospora crassa where its seven chromosomes form a Rabl conformation, where heterochromatic centromeres and telomeres independently cluster at the nuclear membrane, while interspersed heterochromatic loci in Neurospora aggregate across megabases of linear genomic distance for forming TAD-like structures. However, the role of individual heterochromatic loci in normal genome organization and function is unknown. Here, we examined the genome organization of a Neurospora strain harboring a ~41 kilobase facultative (temporarily silent) heterochromatic region deletion, as well as the genome organization of a strain deleted of a ~110 kilobase permanently silent constitutive heterochromatic region. While the facultative heterochromatin deletion had little effect on local chromatin structure, the constitutive heterochromatin deletion altered local TAD-like structures, gene expression, and the predicted 3D genome structure by qualitatively repositioning genes into the nucleus center. Our work elucidates the role of individual heterochromatic regions for genome organization and function.
Project description:Genome organization is essential for proper function, including gene expression. In metazoan genome organization, chromatin loops and Topologically Associated Domains (TADs) facilitate local gene clustering, while chromosomes form distinct nuclear territories characterized by compartmentalization of silent heterochromatin at the nuclear periphery and active euchromatin in the nucleus center. A similar hierarchical organization occurs in the fungus Neurospora crassa where its seven chromosomes form a Rabl conformation, where heterochromatic centromeres and telomeres independently cluster at the nuclear membrane, while interspersed heterochromatic loci in Neurospora aggregate across megabases of linear genomic distance for forming TAD-like structures. However, the role of individual heterochromatic loci in normal genome organization and function is unknown. Here, we examined the genome organization of a Neurospora strain harboring a ~41 kilobase facultative (temporarily silent) heterochromatic region deletion, as well as the genome organization of a strain deleted of a ~110 kilobase permanently silent constitutive heterochromatic region. While the facultative heterochromatin deletion had little effect on local chromatin structure, the constitutive heterochromatin deletion altered local TAD-like structures, gene expression, and the predicted 3D genome structure by qualitatively repositioning genes into the nucleus center. Our work elucidates the role of individual heterochromatic regions for genome organization and function.
Project description:Transcription of genes residing within constitutive heterochromatin is paradoxical to the tenets of epigenetic code. Regulatory mechanisms of Drosophila melanogaster heterochromatic gene transcription remain largely unknown. We investigated the contribution of pericentromeric genome organization and heterochromatic factors in orchestrating heterochromatic gene expression. Using 5C-seq, we characterized the pericentromeric TADs in Drosophila melanogaster. Het TAD borders are enriched in nuclear matrix attachment regions while the intra-TAD interactions are mediated by various insulator binding proteins. Heterochromatic genes of similar expression levels cluster into Het TADs, indicating transcriptional co-regulation. HP1a or Su(var)3-9 RNAi results in perturbation of global pericentromeric TAD organization but the expression of the heterochromatic genes is minimally affected. A subset of active heterochromatic genes has been shown to have combination of HP1a/H3K9me3 with H3K36me3 at their exons. Consequently, knock-down of dMES-4 (H3K36 methyl transferase) downregulates expression of the heterochromatic genes. Furthermore, dADD1, present near the TSS of the active heterochromatic genes, is likely to regulate the heterochromatic gene expression in the presence of HP1a or H3K9me3 marks. Therefore, our findings provide mechanistic insights into the interplay of chromatin interactions and the combination of heterochromatic factors (HP1a, H3K9me3, dMES-4 and dADD1) in regulating heterochromatic gene expression.
Project description:Transcription of genes residing within constitutive heterochromatin is paradoxical to the tenets of epigenetic code. Regulatory mechanisms of Drosophila melanogaster heterochromatic gene transcription remain largely unknown. We investigated the contribution of pericentromeric genome organization and heterochromatic factors in orchestrating heterochromatic gene expression. Using 5C-seq, we characterized the pericentromeric TADs in Drosophila melanogaster. Het TAD borders are enriched in nuclear matrix attachment regions while the intra-TAD interactions are mediated by various insulator binding proteins. Heterochromatic genes of similar expression levels cluster into Het TADs, indicating transcriptional co-regulation. HP1a or Su(var)3-9 RNAi results in perturbation of global pericentromeric TAD organization but the expression of the heterochromatic genes is minimally affected. A subset of active heterochromatic genes has been shown to have combination of HP1a/H3K9me3 with H3K36me3 at their exons. Consequently, knock-down of dMES-4 (H3K36 methyl transferase) downregulates expression of the heterochromatic genes. Furthermore, dADD1, present near the TSS of the active heterochromatic genes, is likely to regulate the heterochromatic gene expression in the presence of HP1a or H3K9me3 marks. Therefore, our findings provide mechanistic insights into the interplay of chromatin interactions and the combination of heterochromatic factors (HP1a, H3K9me3, dMES-4 and dADD1) in regulating heterochromatic gene expression.
Project description:Whether and how histone post-translational modifications and the proteins that bind them drive 3D genome organization remains unanswered. Here, we evaluate the contribution of H3K9-methylated constitutive heterochromatin to 3D genome organization in Drosophila tissues. We find that the predominant organizational feature of wildtype tissues is segregation of euchromatic chromosome arms from heterochromatic pericentromeres. Reciprocal perturbation of HP1a•H3K9me binding, using a point mutation in the HP1a chromodomain or replacement of the replication-dependent histone H3 with H3K9R mutant histones, revealed that HP1a binding to methylated H3K9 in constitutive heterochromatin is required to limit contact frequency between pericentromeres and chromosome arms and regulate the distance between arm and pericentromeric regions. Surprisingly, self-association of pericentromeric regions is largely preserved despite loss of H3K9 methylation and HP1a occupancy. Thus, the HP1a•H3K9 interaction contributes to, but does not solely drive, segregation of euchromatin and heterochromatin inside the nucleus.