ABSTRACT
Heterochromatin is important for gene regulation and chromosome structure, but the genes that are occupied by heterochromatin proteins in the mammalian genome are largely unknown. We have adapted the DamID method to systematically identify target genes of the heterochromatin proteins HP1 and SUV39H1 in human and mouse cells. Unexpectedly, we found that CBX1 (formerly HP1beta) and SUV39H1 bind to genes encoding KRAB domain containing zinc finger (KRAB-ZNF) transcriptional repressors. These genes constitute one of the largest gene families and are organized in clusters in the human genome. Preference of CBX1 for this gene family was observed in both human and mouse cells. High-resolution mapping on human chromosome 19 revealed that CBX1 coats large domains 0.1-4 Mb in size, which coincide with the position of KRAB-ZNF gene clusters. These domains show an intricate CBX1 binding pattern: While CBX1 is globally elevated throughout the domains, it is absent from the promoters and binds more strongly to the 3' ends of KRAB-ZNF genes. KRAB-ZNF domains contain large numbers of LINE elements, which may contribute to CBX1 recruitment. These results uncover a surprising link between heterochromatin and a large family of regulatory genes in mammals. We suggest a role for heterochromatin in the evolution of the KRAB-ZNF gene family.
Subject(s)
DNA-Binding Proteins/genetics , Heterochromatin/chemistry , Protein Structure, Tertiary , Repressor Proteins/genetics , Cell Line , Cell Line, Tumor , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone/chemistry , Chromosome Mapping , Chromosomes, Human, Pair 19 , Gene Expression Profiling , Genome, Human , Humans , Long Interspersed Nucleotide Elements , Methyltransferases/chemistry , Oligonucleotide Array Sequence Analysis , Protamines/chemistry , Protein Binding , Repressor Proteins/chemistryABSTRACT
2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency is a novel inborn error of isoleucine degradation. In this article, we report the elucidation of the molecular basis of MHBD deficiency. To this end, we purified the enzyme from bovine liver. MALDI-TOF mass spectrometry analysis revealed that the purified protein was identical to bovine 3-hydroxyacyl-CoA dehydrogenase type II. The human homolog of this bovine enzyme is a short-chain 3-hydroxyacyl-CoA dehydrogenase, also known as the "endoplasmic reticulum-associated amyloid-beta binding protein" (ERAB). This led to the identification of the X-chromosomal gene involved, which previously had been denoted "HADH2." Sequence analysis of the HADH2 gene from patients with MHBD deficiency revealed the presence of two missense mutations (R130C and L122V). Heterologous expression of the mutant cDNAs in Escherichia coli showed that both mutations almost completely abolish enzyme activity. This confirms that MHBD deficiency is caused by mutations in the HADH2 gene.
