RESUMO
Histones modulate gene expression by chromatin compaction, regulating numerous processes such as differentiation. However, the mechanisms underlying histone degradation remain elusive. Human embryonic stem cells (hESCs) have a unique chromatin architecture characterized by low levels of trimethylated histone H3 at lysine 9 (H3K9me3), a heterochromatin-associated modification. Here we assess the link between the intrinsic epigenetic landscape and ubiquitin-proteasome system of hESCs. We find that hESCs exhibit high expression of the ubiquitin-conjugating enzyme UBE2K. Loss of UBE2K upregulates the trimethyltransferase SETDB1, resulting in H3K9 trimethylation and repression of neurogenic genes during differentiation. Besides H3K9 trimethylation, UBE2K binds histone H3 to induce its polyubiquitination and degradation by the proteasome. Notably, ubc-20, the worm orthologue of UBE2K, also regulates histone H3 levels and H3K9 trimethylation in Caenorhabditis elegans germ cells. Thus, our results indicate that UBE2K crosses evolutionary boundaries to promote histone H3 degradation and reduce H3K9me3 repressive marks in immortal cells.
Assuntos
Histonas/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Neurogênese/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Diferenciação Celular , Epigênese Genética , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Pluripotent stem cells are invaluable resources to study development and disease, holding a great promise for regenerative medicine. Here we use human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) from patients with Huntington's disease (HD-iPSCs) to shed light into the normal function of huntingtin (HTT) and its demise in disease. We find that HTT binds ATF7IP, a regulator of the histone H3 methyltransferase SETDB1. HTT inhibits the interaction of the ATF7IP-SETDB1 complex with other heterochromatin regulators and transcriptional repressors, maintaining low levels of H3K9 trimethylation (H3K9me3) in hESCs. Loss of HTT promotes global increased H3K9me3 levels and enrichment of H3K9me3 marks at distinct genes, including transcriptional regulators of neuronal differentiation. Although these genes are normally expressed at low amounts in hESCs, HTT knockdown (KD) reduces their induction during neural differentiation. Notably, mutant expanded polyglutamine repeats in HTT diminish its interaction with ATF7IP-SETDB1 complex and trigger H3K9me3 in HD-iPSCs. Conversely, KD of ATF7IP in HD-iPSCs reduces H3K9me3 alterations and ameliorates gene expression changes in their neural counterparts. Taken together, our results indicate ATF7IP as a potential target to correct aberrant H3K9me3 levels induced by mutant HTT.
Assuntos
Proteína Huntingtina/genética , Doença de Huntington/genética , Proteínas Metiltransferases/genética , Fatores de Transcrição/genética , Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/patologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Heterocromatina/genética , Histona Metiltransferases/genética , Histona-Lisina N-Metiltransferase , Humanos , Doença de Huntington/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Lentivirus/genética , Neurônios/metabolismo , Neurônios/patologia , Peptídeos/genética , Proteínas RepressorasRESUMO
Much information can be obtained from the chemical composition of a fingermark, which can be helpful in crime scene investigation. Immunolabeling can be used to extract information about the donor of the fingermark and it can also act as a fingermark development tool in sequence with the standard fingermark development techniques. However, before immunolabeling can be used in forensic practice more information on the possibilities and limitations of this technique is required. In this study, our aim was to investigate if immunolabeling is compatible with standard development protocols (indanedione-zinc, indanedione-zinc followed by ninhydrin spraying, physical developer, cyanoacrylate fuming, cyanoacrylate followed by basic yellow staining, lumicyanoacrylate fuming and polycyanoacrylate fuming). Immunolabeling was carried out successfully on all developed fingermarks, whereby dermcidin was selected as antigen of interest. We can conclude that immunolabeling is compatible with a wide variety of different fingermark developers. This finding in combination with previous findings, makes immunolabeling an interesting technique, which can be of great value in the forensic field.
