CLLU1 Expression Is Not Confined to Chronic Lymphocytic Leukemia Cells; Putative Role of Gene-Body Hypomethylation in Regulating Splice Variant-Specific Transcriptional Dynamics

Background: Alternative splicing of the CLLU1 gene generates 6 transcript variants (accession numbers from the National Center for Biotechnology Information: AJ845162-AJ845167), two of which comprise a putative coding DNA sequence (AJ845165 and AJ845166, CDS transcripts). Previous studies showed that CLLU1 overexpression is highly confined to chronic lymphocytic leukemia (CLL) cells and that only the AJ845162 is expressed in both high- and low-risk CLL patients, while expression of the CDS-transcripts is confined to the high-risk subgroup.

Pair-wise linear regression analysis revealed a powerful linear relation between the expression levels of all the splice variants, thereby it was deduced that CLLU1 transcription is orchestrated by a common promoter, which is located upstream of CLLU1 exon 1 (Buhl et al., Leukemia 2009). We recently documented (Papamichos et al., Haematologica 2016; s1) that this promoter was evolutionarily derived by an endogenous retrovirus sequence.

Herein we argue that CLLU1 expression is not restricted to CLL cells while also we highlight that transcription of CDS transcripts is regulated by an alternative intronic promoter.

Methods: CLLU1 high-throughput sequencing of polyadenylated RNA (RNA-Seq) data across 51 human tissues and 2 human cell lines, generated by the Genotype-Tissue Expression (GTEx) project of the US National Institutes of Health Common Fund (GTEx Analysis Release V6p), were analyzed via GTEx Portal. CLLU1 GeneChip expression data (AJ845164 transcript-specific probe set 1558185_at from Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array), generated by Genevestigator project, were downloaded from Genevisible portal.

CLLU1 chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-Seq) on histone H3 protein subunit modified at lysine position 4 with trimethylation (H3K4me3) as well as transcription factor (TF) ChIP-Seq data were downloaded from the Encyclopedia of DNA Elements (ENCODE) Regulation supertrack, which is available in UCSC Genome Browser Database. Data on CLLU1 gene-body methylation status in 63 cell lines from ENCODE were downloaded from the HAIB Methyl450 ENCODE track.

Results: CLLU1 GeneChip expression data show that the AJ845164 transcript (corresponding to the RP11-693J15.4 long non-coding RNA) is promiscuously expressed in various normal B-cell subpopulations andin mature B-cell neoplasms other than CLL, especially in mantle cell lymphoma.RNA-Seq data analysis reveals that AJ845162 transcript is expressed in normal somatic tissues such as skeletal muscle, salivary gland, and esophageal mucosa. Vice versa, expression of CLLU1 CDS transcripts is confined to transformed lymphocytes.

Structure interrogation of CDS transcripts indicates that these variants initiate from an alternative transcriptional start site which is located 2429 nucleotides downstream from the canonical one. H3K4me3 ChIP-Seq data signify the presence of an alternative intronic promoter in this genomic segment. TF Chip-Seq data reveal that the alternative promoter region comprises high affinity binding sites for signal transducer and activator of transcription 3 (STAT3) and Activator protein 1 (AP-1). Analysis of CLLU1 -body methylation status in five lymphoblastoid cell lines reveals that the alternative promoter remains heavily methylated in these cell types. This finding is in accordance with previous studies reporting that CLLU1 gene body is hypermethylated in B-cells from healthy individuals and in IgV_H-mutated (IgV_H-M) CLL cases while appears hypomethylated in IgV_H-unmutated (IgV_H-UM) CLL cells.

Conclusion: Not all CLLU1 transcripts are equal in discriminating CLL from other mature B-cell malignancies.

Whilst of retroviral origin, CLLU1 upstream promoter is not subjected to stringent epigenetic repression but remains active in somatic cells, a tantalizing finding that warrants further investigation.

The level of intragenic DNA methylation represents a decisive regulator of alternative promoter usage (Neri et al., Nature 2017). It would be tantalizing to speculate that CLLU1 -body hypomethylation, occuring exclusively in IgV_H-unmutated CLL, is mechanistically linked to the epigenetic derepression of the alternative intronic CLLU1 promoter and the subsequent high expression of CLLU1 CDS transcripts in high-risk CLL.