Abstract
The human transcription factor DNA replication-related element-binding factor (hDREF) is essential for the transcription of a number of housekeeping genes. The mechanisms underlying constitutively active transcription by hDREF were unclear. Here, we provide evidence that hDREF possesses small ubiquitin-like modifier (SUMO) ligase activity and can specifically SUMOylate Mi2α, an ATP-dependent DNA helicase in the nucleosome remodeling and deacetylation complex. Moreover, immunofluorescent staining and biochemical analyses showed that coexpression of hDREF and SUMO-1 resulted in dissociation of Mi2α from chromatin, whereas a SUMOylation-defective Mi2α mutant remained tightly bound to chromatin. Chromatin immunoprecipitation and quantitative RT-PCR analysis demonstrated that Mi2α expression diminished transcription of the ribosomal protein genes, which are positively regulated by hDREF. In contrast, coexpression of hDREF and SUMO-1 suppressed the transcriptional repression by Mi2α. These data indicate that hDREF might incite transcriptional activation by SUMOylating Mi2α, resulting in the dissociation of Mi2α from the gene loci. We propose a novel mechanism for maintaining constitutively active states of a number of hDREF target genes through SUMOylation.
Highlights
Results human transcription factor DNA replication-related element-binding factor (hDREF) Interacts with Factors Involved in the SUMOylation Pathway and Is SUMOylated Both in Vitro and in Vivo—To define the molecular mechanisms by which hDREF activates transcription, we screened for hDREF-interacting proteins using the yeast two-hybrid system with a human brain cDNA library and full-length hDREF as bait
Both antibodies detected a major 180 kDa band and minor higher migrating bands in samples containing WT small ubiquitin-related modifier (SUMO)-1, the latter of which were absent in lysates from G97A-expressing cells, indicating that these slower migrating bands corresponded to SUMOylated hDREF
This study is the first to demonstrate that the sequence-specific transcription factor hDREF possesses SUMO ligase activity and stimulates transcription of target genes via direct SUMO ligation to Mi2␣, an ATP-dependent nucleosome-remodeling enzyme in the nucleosome remodeling and deacetylation (NuRD) complex
Summary
Cell Culture—HeLa cells were cultured in Ham’s F-12 medium supplemented with 10% FCS, 100 units/ml penicillin, 1 g/ml streptomycin, and 29.2 g/ml L-glutamine at 37 °C under 5% CO2. 293FT cells were maintained in DMEM supplemented with 10% FCS, 0.1 mM non-essential amino acids, 100 units/ml penicillin, 1 g/ml streptomycin, 29.2 g/ml L-glutamine, and 100 g/ml G418 at 37 °C under 5% CO2. For constructing pcDNA3-HAhDREF[332–524] or -(415–524), a cDNA fragment encoding aa residues 332–524 or 415–524, obtained by digesting pcDNA3HA-hDREF[332– 694] or pcDNA3-HA-hDREF[415– 694] with BglII, creating blunt ends with Klenow fragment, and cutting with EcoRI, was ligated between the EcoRI and blunt-ended XhoI sites of pcDNA3-HA. For constructing pcDNA3-HAhDREF[332– 416], a cDNA fragment encoding aa residues 332– 416, obtained by digesting pcDNA3-HA-hDREF[332– 524] with Asp718, creating blunt ends with Klenow fragment, and cutting with EcoRI, was ligated between the EcoRI and blunt-ended XhoI sites of pcDNA3-HA. For constructing phGFP105-hDREF[332– 416], a cDNA fragment encoding aa residues 332– 416, obtained by digesting pcDNA3-HAhDREF[332– 416] with NheI, creating blunt ends with Klenow fragment, and cutting with XhoI, was ligated between the SalI and blunt-ended EcoRI sites of phGFP105-C1.
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