Page 124 - Haematologica May 2022

S. Napoli et al.
non-coding transcripts (e-lncRNA),12 and the stabilization of these eRNA confers to them the capability to act in trans, regulating several distant targets.13
Individual eRNA are expressed in a tissue-specific man- ner. In normal B cells at various stages of differentiation, the expression of non-coding RNA can more precisely define cellular subsets than protein-coding transcripts.14,15 In particular, eRNA are differentially expressed during B- cell development and they are associated with protein- coding genes that play an essential role in B-cell differen- tiation.
Diffuse large B-cell lymphoma (DLBCL) derives from germinal center (GC) B cells. DLBCL is typically divided into two main subtypes: GC B cell-like (GCB-DLBCL), whose transcriptional profile resembles that of light zone GC B cells, and activated B cell-like (ABC-DLBCL), whose transcriptome resembles that of plasmablasts.16 However, DLBCL within each of these subgroups exhibit biological, genetic and transcriptional heterogeneity.17-19 Lineage-spe- cific and growth-dependent transcription factors like BCL6, Myc, NF-kB, p53, and E2F1 can activate specific genetic signatures, depending on the activation of unique subsets of enhancers20,21 and contribute to disease hetero- geneity. Here, we studied a unique eRNA associated with the POU2AF1 gene, that we termed GECPAR, for germi- nal center proliferative adapter RNA. POU2AF1 encodes the protein OCA-B, co-activator of OCT2, a B-cell specif- ic transcription factor which plays a pivotal role in the regulation of normal and neoplastic GC B cells.22,23 The SE proximal to POU2AF1 is the most activated SE in GCB- DLBCL.23 Loss of GECPAR correlated with reduced tran- scription of TLE4, which is a negative regulator of LEF1, a Wnt pathway effector protein that in turn regulates also NF-kB. GECPAR loss also increased MYC expression and proliferation of DLBCL cell lines. Conversely, its overex- pression impaired cell proliferation. Collectively, our data provides evidence of the nodal role of GECPAR in the reg- ulatory network modulating B-cell differentiation and proliferation.
Methods
Detailed descriptions of the experimental methods are includ- ed in the Online Supplementary Appendix.
Human samples, cell lines, small interfering RNA transfection
Established human DLBCL cell lines and patient-derived tumor xenograft cell lines (PDTX-CL) were grown as previously described.24 All patients providing samples gave written informed consent. Molecular and clinical data acquisition and PDTX estab- lishment were approved and carried out in accordance with Declaration of Helsinki and were approved by Institutional Review Boards of the New York Presbyterian Hospital, Weill Cornell Medicine (WCM) and the Ospedale San Giovanni Battista delle Molinette. Cell lines were checked for their identity.24 Cells were transfected with small interfering RNA (siRNA) or locked- nucleic acid (LNA) using the 4D Nucleofector.
GECPAR cloning and infection into lymphoma cells, RNA sequencing
Cellular lysates were fractionated as previously described.25 For strand-specific quantitative reverse transcription polymerase chain reaction (qRT-PCR), only the forward primer was used to
amplify the antisense strand and only the reverse primer to amplify the sense strand. 5’ and 3’ rapid amplification of cDNA ends (RACE) was done using Invitrogen RACE System kits. GECPAR was cloned into the pGEM-T vector and subcloned in pCDH-CMV-MCS-EF1-copGFP. pCDH empty backbone or pCDH_GECPAR were transfected in HEK293T, and viral super- natant was then used to infect lymphoma cells. RNA sequencing (RNA-Seq) in cell lines was performed using the NEBNext Ultra II Directional RNA Library Prep.
Capture hybridization analysis of RNA targets (CHART) sequencing
CHART enrichment and ribonuclease H (RNAseH) mapping experiments were performed following previously reported pro- tocols.26,27 The enrichment of CHART signals was determined relative to the oligo controls. Conservative enrichment profiles were determined using the SPP package28 and MACS,29 as described by Vance and colleagues.30
Results
The super-enhancer associated with the POU2AF1 gene locus is transcribed in normal B cells and diffuse large B-cell lymphoma cell lines
Analysis of publicly available RNA-Seq data on RNA polyA+ or polyA-31 showed that CD20+ cells express a non-polyadenylated portion of the LOC100132078 tran- script and also two isoforms of a more abundant anti- sense transcript (Figure 1A; Online Supplementary Figure S1A). Due to its proximity to the POU2AF1 gene and its localization in a genomic region with characteristic SE features (highly acetylated, enriched in H3K4me1 but not H3K4me3, based on ENCODE ChIP-Seq data), we hypothesized that it could be an eRNA with particular relevance for GC B cells
In order to confirm the eRNA length reconstructed in CD20+ cells, we performed 5’ and 3’ RACE in the DLBCL cell line OCI-LY1. For the 3’-end detection we ran two reactions, with or without the addition of an artificial polyA tail. We identified a transcript lacking a polyA tail and another that was 400 bases longer and naturally polyadenylated. Similarly to the aforementioned polyA- transcript reported in CD20+ normal B cells, neither of the transcripts identified in DLBCL cells extended beyond the annotated first exon. The 5’ RACE reaction reverse transcribed from exon 4 did not identify a specific 5’-end for exon 1, indicating that the long annotated transcript, LOC100132078, was likely not stable in our model. Conversely, reverse transcribing from exon 1, we identi- fied a 5’-end located at nucleotide +366, mirroring our in silico observations for CD20+ normal B cells (Figure 1A and B). We renamed the stabilized portion of LOC100132078 we had sequenced in the OCI-LY1 model as “GECPAR”.
GECPAR is mainly chromatin associated and partially polyadenylated
In order to further characterize the physical characteris- tics of GECPAR, RNA was extracted from the cytoplasm, nucleoplasm and chromatin fractions. In GCB-DLBCL (OCI-LY1 and Karpas422) and ABC-DLBCL (HBL1, U2932) cell lines, GECPAR was transcribed but mostly retained on chromatin, in accordance with reported fea- tures of eRNA.6,7 It was also clearly detected in the nucle-
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