RESUMO
The discovery of biased histone inheritance in asymmetrically dividing Drosophila melanogaster male germline stem cells demonstrates one means to produce two distinct daughter cells with identical genetic material. This inspired further studies in different systems, which revealed that this phenomenon may be a widespread mechanism to introduce cellular diversity. While the extent of asymmetric histone inheritance could vary among systems, this phenomenon is proposed to occur in three steps: first, establishment of histone asymmetry between sister chromatids during DNA replication; second, recognition of sister chromatids carrying asymmetric histone information during mitosis; and third, execution of this asymmetry in the resulting daughter cells. By compiling the current knowledge from diverse eukaryotic systems, this review comprehensively details and compares known chromatin factors, mitotic machinery components, and cell cycle regulators that may contribute to each of these three steps. Also discussed are potential mechanisms that introduce and regulate variable histone inheritance modes and how these different modes may contribute to cell fate decisions in multicellular organisms.
Assuntos
Drosophila melanogaster , Histonas , Animais , Histonas/genética , Drosophila melanogaster/genética , Padrões de Herança , Cromatina/genética , CromátidesRESUMO
Adult stem cells undergo asymmetric cell divisions to produce 2 daughter cells with distinct cell fates: one capable of self-renewal and the other committed for differentiation. Misregulation of this delicate balance can lead to cancer and tissue degeneration. During asymmetric division of Drosophila male germline stem cells (GSCs), preexisting (old) and newly synthesized histone H3 are differentially segregated, whereas old and new histone variant H3.3 are more equally inherited. However, what underlies these distinct inheritance patterns remains unknown. Here, we report that the N-terminal tails of H3 and H3.3 are critical for their inheritance patterns, as well as GSC maintenance and proper differentiation. H3 and H3.3 differ at the 31st position in their N-termini with Alanine for H3 and Serine for H3.3. By swapping these 2 amino acids, we generated 2 mutant histones (i.e., H3A31S and H3.3S31A). Upon expressing them in the early-stage germline, we identified opposing phenotypes: overpopulation of early-stage germ cells in the H3A31S-expressing testes and significant germ cell loss in testes expressing the H3.3S31A. Asymmetric H3 inheritance is disrupted in the H3A31S-expressing GSCs, due to misincorporation of old histones between sister chromatids during DNA replication. Furthermore, H3.3S31A mutation accelerates old histone turnover in the GSCs. Finally, using a modified Chromatin Immunocleavage assay on early-stage germ cells, we found that H3A31S has enhanced occupancy at promoters and transcription starting sites compared with H3, while H3.3S31A is more enriched at transcriptionally silent intergenic regions compared to H3.3. Overall, these results suggest that the 31st amino acids for both H3 and H3.3 are critical for their proper genomic occupancy and function. Together, our findings indicate a critical role for the different amino acid composition of the N-terminal tails between H3 and H3.3 in an endogenous stem cell lineage and provide insights into the importance of proper histone inheritance in specifying cell fates and regulating cellular differentiation.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Histonas/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Linhagem da Célula/genética , Células Germinativas/metabolismo , Aminoácidos/metabolismoRESUMO
The conserved histone locus body (HLB) assembles prior to zygotic gene activation early during development and concentrates factors into a nuclear domain of coordinated histone gene regulation. Although HLBs form specifically at replication-dependent histone loci, the cis and trans factors that target HLB components to histone genes remained unknown. Here we report that conserved GA repeat cis elements within the bidirectional histone3-histone4 promoter direct HLB formation in Drosophila In addition, the CLAMP (chromatin-linked adaptor for male-specific lethal [MSL] proteins) zinc finger protein binds these GA repeat motifs, increases chromatin accessibility, enhances histone gene transcription, and promotes HLB formation. We demonstrated previously that CLAMP also promotes the formation of another domain of coordinated gene regulation: the dosage-compensated male X chromosome. Therefore, CLAMP binding to GA repeat motifs promotes the formation of two distinct domains of coordinated gene activation located at different places in the genome.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos , Histonas/genética , Animais , Sequência de Bases , Cromatina/metabolismo , Sequência Conservada , DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Histonas/metabolismo , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Fatores de Transcrição/metabolismoRESUMO
Heterogametic species require chromosome-wide gene regulation to compensate for differences in sex chromosome gene dosage. In Drosophila melanogaster, transcriptional output from the single male X-chromosome is equalized to that of XX females by recruitment of the male-specific lethal (MSL) complex, which increases transcript levels of active genes 2-fold. The MSL complex contains several protein components and two non-coding RNA on the X ( roX) RNAs that are transcriptionally activated by the MSL complex. We previously discovered that targeting of the MSL complex to the X-chromosome is dependent on the chromatin-linked adapter for MSL proteins (CLAMP) zinc finger protein. To better understand CLAMP function, we used the CRISPR/Cas9 genome editing system to generate a frameshift mutation in the clamp gene that eliminates expression of the CLAMP protein. We found that clamp null females die at the third instar larval stage, while almost all clamp null males die at earlier developmental stages. Moreover, we found that in clamp null females roX gene expression is activated, whereas in clamp null males roX gene expression is reduced. Therefore, CLAMP regulates roX abundance in a sex-specific manner. Our results provide new insights into sex-specific gene regulation by an essential transcription factor.
Assuntos
Proteínas de Ligação a DNA/genética , Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Proteínas Nucleares/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Cromossomo X/genética , Animais , Proteínas de Ligação a DNA/fisiologia , Feminino , Masculino , RNA não TraduzidoRESUMO
Gaining new insights into gene regulation involves an in-depth understanding of protein-protein interactions on chromatin. A powerful model for studying mechanisms of gene regulation is dosage compensation, a process that targets the X-chromosome to equalize gene expression between XY males and XX females. We previously identified a zinc finger protein in Drosophila melanogaster that plays a sex-specific role in targeting the Male-specific lethal (MSL) dosage compensation complex to the male X-chromosome, called the Chromatin-Linked Adapter for MSL Proteins (CLAMP). More recently, we established that CLAMP has non-sex-specific roles as an essential protein that regulates chromatin accessibility at promoters genome-wide. To identify associations between CLAMP and other factors in both male and female cells, we used two complementary mass spectrometry approaches. We demonstrate that CLAMP associates with the transcriptional regulator complex Negative Elongation Factor (NELF) in both sexes and determine that CLAMP reduces NELF recruitment to several target genes. In sum, we have identified many new CLAMP-associated factors and provide a resource for further study of this little understood essential protein.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Animais , Sítios de Ligação , Feminino , Imunoprecipitação , Masculino , Ligação Proteica , Fatores de Transcrição/metabolismoRESUMO
Chromatin accessibility plays a fundamental role in gene regulation. Nucleosome placement, usually measured by quantifying protection of DNA from enzymatic digestion, can regulate accessibility. We introduce a metric that uses micrococcal nuclease (MNase) digestion in a novel manner to measure chromatin accessibility by combining information from several digests of increasing depths. This metric, MACC (MNase accessibility), quantifies the inherent heterogeneity of nucleosome accessibility in which some nucleosomes are seen preferentially at high MNase and some at low MNase. MACC interrogates each genomic locus, measuring both nucleosome location and accessibility in the same assay. MACC can be performed either with or without a histone immunoprecipitation step, and thereby compares histone and non-histone protection. We find that changes in accessibility at enhancers, promoters and other regulatory regions do not correlate with changes in nucleosome occupancy. Moreover, high nucleosome occupancy does not necessarily preclude high accessibility, which reveals novel principles of chromatin regulation.