Role of Helicase-Like Transcription Factor (Hltf) in the G2/M Transition and Apoptosis in Brain

Abstract

HLTF participates in transcription, chromatin remodeling, DNA damage repair, and tumor suppression. Aside from being expressed in mouse brain during embryonic and postnatal development, little is known about Hltf's functional importance. Splice variant quantification of wild-type neonatal (6-8 hour postpartum) brain gave a ratio of 5:1 for Hltf isoform 1 (exons 1-25) to isoform 2 (exons 1-21 with exon 21 extended via a partial intron retention event). Western analysis showed a close correlation between mRNA and protein expression. Complete loss of Hltf caused encephalomalacia with increased apoptosis, and reduced viability. Sixty-four percent of Hltf null mice died, 48% within 12-24 hours of birth. An RNA-Seq snapshot of the neonatal brain transcriptome showed 341 of 20,000 transcripts were altered (p < 0.05) - 95 up regulated and 246 down regulated. MetaCoreTM enrichment pathway analysis revealed Hltf regulates cell cycle, cell adhesion, and TGF-beta receptor signaling. Hltf's most important role is in the G2/M transition of the cell cycle (p = 4.672e-7) with an emphasis on transcript availability of major components in chromosome cohesion and condensation. Hltf null brains have reduced transcript levels for Rad21/Scc1, histone H3.3, Cap-E/Smc2, Cap-G/G2, and Aurora B kinase. The loss of Hltf in its yeast Rad5-like role in DNA damage repair is accompanied by down regulation of Cflar, a critical inhibitor of TNFRSF6-mediated apoptosis, and increased (p<0.0001) active caspase-3, an indicator of intrinsic triggering of apoptosis in null brains. Hltf also regulates Smad7/Bambi/Tgf-beta/Bmp5/Wnt10b signaling in brain. ChIP confirmed Hltf binding to consensus sequences in predicted (promoter Scgb3a1 gene) and previously unidentified (P-element on chromosome 7) targets. This study is the first to provide a comprehensive view of Hltf targets in brain. Moreover, it reveals how silencing Hltf disrupts cell cycle progression, and attenuates DNA damage repair.

The helicase-like transcription factor (HLTF) in cancer: loss of function or oncomorphic conversion of a tumor suppressor?

“…Qing et al [19] demonstrated that Ubiquitin Specific Peptidase 7 (USP7), a deubiquination enzyme (DUB), interacts with and stabilizes HLTF both in vitro and in vivo. In USP7-depleted cells, the levels of HLTF protein, but not mRNA, [23]). Above each protein, the mRNA area of exons 19-22 is represented.…”

“…Two different consensus sequences recognized by HLTF were proposed: (A/G)G(T/C)(G/T)G [9] and (C/A)C(T/A)TN(T/G) [6]. The latter sequence was successfully used in a Genomatix analysis (MatInspector; [22]) to develop an algorithm for identifying putative HLTF binding sites in genomic sequences ( [23]; Table 1). The two HLTF binding consensus sequences were defined using a similar screening strategy from a collection of random sequence DNA oligonucleotides: a target definition assay [32] or CASTing [33].…”

“…The only difference was that the screening was performed using either the HLTF DNA-binding domain expressed in E. coli as a glutathione-S-transferase (GST) fusion [32] or HLTF in rabbit nuclear extracts [33]. HLTF binding to these sequences was confirmed through sitedirected mutagenesis and luciferase assays [32] and through chromatin immunoprecipitation (ChIP; [21,23,25,27,[33][34][35][36]). …”

Helicase-like transcription factor is a RUNX1 target whose downregulation promotes genomic instability and correlates with complex cytogenetic features in acute myeloid leukemia

“…Previous studies have revealed that the mRNA expression of cohesin and condensin genes is downregulated in Hltf-deficient cells, 38 suggesting that HLTF may regulate these genes at the transcriptional level. The consensus HLTF DNA-binding sequence has been reported to be 5'MCWTDK3'.…”

“…ATM and ATR are two major kinases that phosphorylate H2AX, 24 and interestingly, their expressions are found to be reduced in Hltfdepleted cells. 40 Of note, the cohesin and condensin complexes are also strongly implicated in other processes including DNA repair and gene regulation, indicating that HLTF deregulation has diverse effects on genome functions. It could be postulated that the high-level HLTF expression in normal human CD34 + cells is required for HSC functions by preventing genomic instability and malignant transformation.…”