Transcription Impairment and Cell Migration Defects in Elongator-Depleted Cells: Implication for Familial Dysautonomia

Abstract

Mutations in IKBKAP, encoding a subunit of Elongator, cause familial dysautonomia (FD), a severe neurodevelopmental disease with complex clinical characteristics. Elongator was previously linked not only with transcriptional elongation and histone acetylation but also with other cellular processes. Here, we used RNA interference (RNAi) and fibroblasts from FD patients to identify Elongator target genes and study the role of Elongator in transcription. Strikingly, whereas Elongator is recruited to both target and nontarget genes, only target genes display histone H3 hypoacetylation and progressively lower RNAPII density through the coding region in FD cells. Interestingly, several target genes encode proteins implicated in cell motility. Indeed, characterization of IKAP/hELP1 RNAi cells, FD fibroblasts, and neuronal cell-derived cells uncovered defects in this cellular function upon Elongator depletion. These results indicate that defects in Elongator function affect transcriptional elongation of several genes and that the ensuing cell motility deficiencies may underlie the neuropathology of FD patients.

Deregulated expression of pro-survival and pro-apoptotic p53-dependent genes upon Elongator deficiency in colon cancer cells

“…Moreover, Elp3, the catalytic subunit, harbours motifs found in the GNAT family of histone acetyltransferases (HATs) [5] and is essential for the ability of Elongator to target histone H3 in vitro [2,3,6] and in vivo [7]. These observations combined with reports showing an association of Elongator with several nascent RNAs in yeast [8] and with the preferential recruitment of Elongator to the transcribed regions of human genes [9][10][11], strongly support a role for Elongator in transcriptional elongation. Meanwhile, other reports also provided experimental evidence for a role of Elongator in exocytosis and tRNA modification [12][13][14].…”

“…Cells were lyzed 48 h post-transfection, and anti-IKAP/hELP1 and -ELP3 Western blots were subsequently performed. The pLL3.7 shRNA IKAP 1 and GFP lentiviral constructs were previously described [11] whereas the shRNA IKAP 2 construct was generated by subcloning another IKAP sequence (available upon request) into the pLL3.7 vector. Lentiviral IKAP/ hELP1 and GFP shRNAs constructs were also generated by subcloning the corresponding sequences into the BLOCK-iT lentiviral RNAi expression system, according to the protocol provided by the manufacturer (Invitrogen, Carlsbad, CA, USA).…”

“…Primer sequences for p21 [24], B2M and IKAP/ hELP1 [11] were previously described and are available upon request, as are the primer sequences for SGK, TNFRSF10D, SESN2, BTG2, PKIB and BAX.…”

“…FD is due to a mutation in a splice site of the IKBKAP gene, which ultimately causes decreased expression of IKAP/hELP1 in a tissue-specific manner [19,20]. Although the molecular and cellular mechanisms underlying this neuro-developmental and neurodegenerative genetic disorder remain largely undefined, impaired cell motility may account for this disease as a variety of human cells depleted for IKAP/hELP1 harboured defects in cell migration [11]. Thus, these studies collectively demonstrate that impaired Elongator function causes multiple cellular defects.…”

“…The monoclonal anti-IKAP/hELP1 and -PARP antibodies were purchased from BD Biosciences Pharmingen (San Jose, CA, USA) whereas the anti-Elp3 antibody was previously described [11]. The anti-p21 and -BAX antibodies were from Calbiochem (San Diego, CA, USA) whereas the anti-serine 15 phospho p53 ( p p53), -p53, -FACT, -CDK9 and -a-tubulin antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA).…”