Project description:We determined the effects of short pulses of DUX4 expression on genome-wide transcription, mimicking the embryonic expression of this gene, using a DUX4-inducible cell culture model of FSHD (MB135iDUX4). We found that a second pulse of DUX4 has a synergistic effect and shows greater induction of DUX4 targets. Knockdown of DUX4-induced histone variants H3.X and H3.Y has no effect on DUX4 targets with a single pulse of DUX4 but eliminates the superinduction seen with a second pulse, showing that these histones are required for this increase in expression.
Project description:We determined the effects of short pulses of DUX4 expression on genome-wide transcription, mimicking the embryonic expression of this gene, using a DUX4-inducible cell culture model of FSHD (MB135iDUX4). We found that a second pulse of DUX4 has a synergistic effect and shows greater induction of DUX4 targets. Knockdown of DUX4-induced histone variants H3.X and H3.Y has no effect on DUX4 targets with a single pulse of DUX4 but eliminates the superinduction seen with a second pulse, showing that these histones are required for this increase in expression.
Project description:The selective incorporation of appropriate histone variants into chromatin is critical for the regulation of genome function. Although many histone variants have been identified, a complete list has not been compiled. We screened human and mouse genomes by in silico hybridization using canonical histone sequences, and found 14 uncharacterized H3-related genes in mouse. Most were similar to the H3.3 variant; their 3′ untranslated regions lacked a stem-loop structure but did have a poly- adenylation signal. Transcriptome analysis revealed tissue-specific expression of these variants. When expressed as GFP-tagged versions in mouse C2C12 cells, some variants were stably incorporated into chromatin and the genome distributions of most variants were similar to that of H3.3. Over-expression of some variants altered gene expression patterns in differentiated C2C12 cells, suggesting that these new H3 variants have specific and distinct functions.
Project description:Nucleosomal incorporation of specialized histone variants is an important mechanism to generate different functional chromatin states. Here we report the identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y. Their mRNAs are found in certain human cell lines, in addition to several normal and malignant human tissues. In keeping with their primate-specificity, H3.X and H3.Y are detected in different brain regions. Transgenic H3.X and H3.Y proteins are stably incorporated into chromatin in a similar fashion to the known H3 variants. Importantly, we demonstrate biochemically and by mass spectrometry that endogenous posttranslationally modified H3.Y protein exists in vivo, and that stress-stimuli, such as starvation and cellular density, increase the abundance of H3.Y-expressing cells. Global transcriptome analysis revealed that knock-down of H3.Y affects cell growth and leads to changes in the expression of many genes involved in cell cycle control. Thus, H3.Y is a novel histone variant involved in the regulation of cellular responses to outside stimuli. Total RNA samples from human U2OS cells. Transcript levels after luciferase, H3.X and/or H3.Y RNAi was analyzed.
Project description:Nucleosomal incorporation of specialized histone variants is an important mechanism to generate different functional chromatin states. Here we report the identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y. Their mRNAs are found in certain human cell lines, in addition to several normal and malignant human tissues. In keeping with their primate-specificity, H3.X and H3.Y are detected in different brain regions. Transgenic H3.X and H3.Y proteins are stably incorporated into chromatin in a similar fashion to the known H3 variants. Importantly, we demonstrate biochemically and by mass spectrometry that endogenous posttranslationally modified H3.Y protein exists in vivo, and that stress-stimuli, such as starvation and cellular density, increase the abundance of H3.Y-expressing cells. Global transcriptome analysis revealed that knock-down of H3.Y affects cell growth and leads to changes in the expression of many genes involved in cell cycle control. Thus, H3.Y is a novel histone variant involved in the regulation of cellular responses to outside stimuli.