Regulation of the cohesin-loading factor NIPBL: Role of the lncRNA NIPBL-AS1 and identification of a distal enhancer element

Abstract

Cohesin is crucial for genome stability, cell division, transcription and chromatin organization. Its functions critically depend on NIPBL, the cohesin-loader protein that is found to be mutated in >60% of the cases of Cornelia de Lange syndrome (CdLS). Other mutations are described in the cohesin subunits SMC1A, RAD21, SMC3 and the HDAC8 protein. In 25-30% of CdLS cases no mutation in the known CdLS genes is detected. Until now, functional elements in the noncoding genome were not characterized in the molecular etiology of CdLS and therefore are excluded from mutation screening, although the impact of such mutations has now been recognized for a wide range of diseases. We have identified different elements of the noncoding genome involved in regulation of the NIPBL gene. NIPBL-AS1 is a long non-coding RNA transcribed upstream and antisense to NIPBL. By knockdown and transcription blocking experiments, we could show that not the NIPBL-AS1 gene product, but its actual transcription is important to regulate NIPBL expression levels. This reveals a possibility to boost the transcriptional activity of the NIPBL gene by interfering with the NIPBL-AS1 lncRNA.Further, we have identified a novel distal enhancer regulating both NIPBL and NIPBL-AS1. Deletion of the enhancer using CRISPR genome editing in HEK293T cells reduces expression of NIPBL, NIPBL-AS1 as well as genes found to be dysregulated in CdLS. Author summaryThe most frequent mutations in the human developmental disorder Cornelia de Lange Syndrome (CdLS) occur in the NIPBL gene. NIPBL is critical for chromatin-association of the cohesin complex and has a dual role as transcription factor. The regulation of the NIPBL gene is of great interest since organisms are very sensitive to NIPBL levels. For instance, severely affected patients showed only~65% and mildly affected patients~75% of NIPBL mRNA levels. One case reports a CdLS phenotype with as little as 15% reduction of the NIPBL transcript. Further, in a number of patients with CdLS phenotype no mutations in known CdLS genes are found, raising the question whether mutations could also occur in gene-regulatory elements. Here we investigate the role of a long non-coding RNA NIPBL-AS1 originating from a promoter shared with NIPBL and observe a co-regulation by a distal enhancer that we have identified and that seems to be conserved between tissues. Deletion of the enhancer by CRISPR leads to reduced expression of NIPBL and NIPBL-AS1 but also of NIPBL target genes that were found to be dysregulated in CdLS patient cells. The lncRNA NIPBL-AS1 itself has no role for NIPBL expression, but its transcription reduces the expression of the NIPBL gene and vice versa, thus revealing an interesting mechanism for the fine-tuning of expression levels.

MAU2 and NIPBL variants in Cornelia de Lange syndrome reveal MAU2-independent loading of cohesin and uncover a protective mechanism against early truncating mutations in NIPBL

“…Altogether, these results corroborate the hypothesis that the NIPBL c.39dup transcripts do not undergo nonsense-mediated mRNA decay in NIPBLΔN cells and, instead, suggest the existence of a feedback mechanism between NIPBL protein and transcript to fine-tune its expression in order to ensure proper execution of its functions. A similar compensation mechanism is also observed in cell lines of patients with mutations in NIPBL, where the wild type allele is frequently upregulated, presumably in order to compensate for the downregulation of the mutant transcript induced by nonsense-mediated mRNA decay 28,33 .…”

“…NIPBL expression was assessed with two different antibodies, one raised against its N-terminus and one recognizing its C-terminus. Notably, mutant NIPBL transcripts partially Page 6 on 47 undergo nonsense-mediated mRNA decay in cell lines of patients with heterozygous truncating variants in NIPBL; consequently, the corresponding truncated protein is unlikely to be detected 32,33 .…”

Identification of co‑expression modules and hub genes of retinoblastoma via co‑expression analysis and protein‑protein interaction networks

“…Similarly, Go analysis in the present study indicated enrichment of the genes in the yellow module in synaptic guidance. among the genes in the turquoise module, niPBl plays a critical and regulatory role in cohesion loading on chromatin, as well as having roles in gene expression and transcriptional signaling (33,34). Zinc finger protein 609 may participate in the regulation of cortical neuron migration (35).…”

Pan-tissue transcriptome analysis of long noncoding RNAs in the American beaver Castor canadensis

“…The relatively large proportion (12 out of 40) of antisense RNAs is consistent with a previous report that antisense transcripts are highly prevalent in the human genome [29]. The list of 16 lncRNA genes includes beaver orthologs for well-known lncRNAs such as XIST [2] (which was the longest of 187 high-confidence lncRNA contigs at 3967 nt), maternally expressed gene 3 (MEG3) [30], terminal differentiation-induced noncoding RNA (TINCR) [31], and nipped-B homolog (Drosophila) long noncoding RNA bidirectional promoter (NIPBL-DT) [32].…”