Project description:Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/ non-specific DNA binding affinity. How these properties affect the ability of TFs to occupy their specific binding sites in the genome and modify the epigenetic landscape is unclear. Here we combined live cell quantitative measurements of mitotic chromosome binding (MCB) of 502 TFs, measurements of TF mobility by fluorescence recovery after photobleaching, single molecule imaging of DNA binding in live cells, and genome-wide mapping of TF binding and chromatin accessibility. MCB scaled with interphase properties such as association with DNA-rich compartments, mobility, as well as large differences in genome-wide specific site occupancy that correlated with TF impact on chromatin accessibility. As MCB is largely mediated by electrostatic, non-specific TF-DNA interactions, our data suggests that non-specific DNA binding of TFs enhances their search for specific sites and thereby their impact on the accessible chromatin landscape.
Project description:Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/ non-specific DNA binding affinity. How these properties affect the ability of TFs to occupy their specific binding sites in the genome and modify the epigenetic landscape is unclear. Here we combined live cell quantitative measurements of mitotic chromosome binding (MCB) of 502 TFs, measurements of TF mobility by fluorescence recovery after photobleaching, single molecule imaging of DNA binding in live cells, and genome-wide mapping of TF binding and chromatin accessibility. MCB scaled with interphase properties such as association with DNA-rich compartments, mobility, as well as large differences in genome-wide specific site occupancy that correlated with TF impact on chromatin accessibility. As MCB is largely mediated by electrostatic, non-specific TF-DNA interactions, our data suggests that non-specific DNA binding of TFs enhances their search for specific sites and thereby their impact on the accessible chromatin landscape.
Project description:Mixed Lineage Leukemia (MLL) and its metazoan Trithorax orthologs have been linked with the epigenetic maintenance of transcriptional activity. To identify mechanisms by which MLL perpetuates active transcription in dividing cells, we investigated its role during M-phase of the cell cycle. Unlike other chromatin modifying enzymes examined, we found that MLL associates with gene promoters packaged within condensed mitotic chromosomes. Genome-wide location analysis identified a globally rearranged pattern of MLL occupancy during mitosis in a manner favoring genes that were highly transcribed during interphase. Knockdown experiments revealed that MLL retention at gene promoters during mitosis accelerates transcription reactivation following mitotic exit. MLL tethers Menin, RbBP5, and ASH2L to its occupied sites during mitosis, but is dispensable for preserving histone H3K4 methylation. These findings implicate mitotic bookmarking as a component of Trithorax-based gene regulation which may facilitate inheritance of active gene expression states during cell division. anti-MLL ChIP (antibody 456) and anti-pol2 chip (sc-899) in chromatin prepared from interphase and mitotic HeLa cells
Project description:During open mitosis chromatin condenses to form chromosomes and decondenses post-mitotically to re-occupy their designated nuclear territory in a precisely lineage specific manner. This necessitates that features of nuclear architecture persist through mitosis. Here we present a proteomic study, which shows that the features of nuclear architecture in interphase nucleus are retained on the mitotic chromosome. Proteomic comparison was made between nuclease and high salt resistant fraction of interphase nucleus known as nuclear matrix (NuMat) and an identical biochemical fraction in the mitotic chromosome known as mitotic chromosome scaffold (MiCS). Our study elucidates that a large graction of the NuMat proteins are retained in the MiCS and possibly play an important role in maintenance of cell lineage specific features of nuclear architecture during cell division and thereby serve as components of cellular memory.
Project description:Phosphorylation of OSBL8 human protein was compared in interphase vs mitotic Hela cells with a new software developed in-house, Msviz, a graphical software tool developed for in-depth manual validation and quantitation of post-translational modifications.
Project description:Mixed Lineage Leukemia (MLL) and its metazoan Trithorax orthologs have been linked with the epigenetic maintenance of transcriptional activity. To identify mechanisms by which MLL perpetuates active transcription in dividing cells, we investigated its role during M-phase of the cell cycle. Unlike other chromatin modifying enzymes examined, we found that MLL associates with gene promoters packaged within condensed mitotic chromosomes. Genome-wide location analysis identified a globally rearranged pattern of MLL occupancy during mitosis in a manner favoring genes that were highly transcribed during interphase. Knockdown experiments revealed that MLL retention at gene promoters during mitosis accelerates transcription reactivation following mitotic exit. MLL tethers Menin, RbBP5, and ASH2L to its occupied sites during mitosis, but is dispensable for preserving histone H3K4 methylation. These findings implicate mitotic bookmarking as a component of Trithorax-based gene regulation which may facilitate inheritance of active gene expression states during cell division.