BioProject

Interstitial deletion of a single copy of chromosome 5q is the most frequent cytogenetic alteration in Myelodysplastic Syndromes (MDS), which results in reduced dosage of numerous genes. More...

Interstitial deletion of a single copy of chromosome 5q is the most frequent cytogenetic alteration in Myelodysplastic Syndromes (MDS), which results in reduced dosage of numerous genes. Furthermore, the extent of the 5q deletion determines disease severity, suggesting cooperation between deleted genes in the proximal and distal regions of del(5q). Although the contribution of individual genes to the pathogenesis of del(5q) MDS has been investigated, less is known about the epistatic interactions and/or cooperation between neighboring deleted genes. Deletion of TRAF-interacting protein with forkhead-associated domain B (TIFAB) and miR-146a, two haploinsufficient genes in del(5q) MDS, has been previously reported to activate the Toll-like receptor (TLR) signaling cascade in hematopoietic stem/progenitor cells (HSPC) by increasing TRAF6 protein stability and mRNA translation, respectively. To investigate the epistasis of TIFAB and miR-146a, we generated a mouse model in which Tifab and miR-146a were simultaneously deleted (Tifab-/-;miR-146a-/-, dKO). Herein, we report that combined hematopoietic-specific deletion of Tifab and miR-146a results in more rapid and severe cytopenia, and progression to a fatal bone marrow (BM) failure-like disease as compared to Tifab- or miR-146adeficiency alone. HSPC from Tifab-/-, miR-146a-/-, and dKO mice exhibit enrichment of gene 69 regulatory networks associated with innate immune signaling. Moreover, a subset of the differentially expressed genes is controlled synergistically following deletion of Tifab and miR-146a. Notably, nearly half of these defined synergy response genes identified in the mouse models were aberrantly expressed in del(5q) MDS HSPC when TIFAB (5q31) and miR-146a (5q33.3) were both deleted. Thus, synergistic control of gene expression following deletion of epistatic haploinsufficient genes in del(5q) MDS may be an underlying mechanism of the diseased state. Overall design: To determine whether combined deletion of Tifab and miR-146a exaggerates the hematopoietic-specific defects observed in either Tifab- or miR-146a-deficient mice in vivo, bone marrow (BM) cells from WT, Tifab-/-, miR-146a-/-, and dKO mice were transplanted into lethally-irradiated syngeneic recipient mice. All recipient mice efficiently engrafted donor BM cells. To gain insight into the molecular nature and collaborative interplay between TIFAB and miR-146a deficiencies responsible for the exaggerated hematopoietic defect associated with MDS, we examined the gene regulatory networks in HSPC from mice transplanted with WT, Tifab-/-, miR- 146a-/-, and dKO BM. The lineage-Sca-1-c-kit+ (LK) BM fractions were analyzed using whole-genome RNA sequencing. Total RNA was amplified using the Ovation RNA-Seq System v2 (NuGEN) according to the manufacturer’s protocol. The libraries were prepared with the Nextera XT DNA Sample Preparation kit (Illumina Technologies). 1 ng of cDNA was suspended in Tagment DNA Buffer, and tagmentation (fragmentation and tagging with the adaptors) was performed with the Nextera enzyme (Amplicon Tagment Mix), incubating at 55C for 10 min. NT Buffer was then added to neutralize the samples. Libraries were prepared by PCR with the Nextera PCR Master Mix, and 2 Nextera Indexes (N7XX, and N5XX) according to the following program: one cycle of 72C for 3min, one cycle of 98C for 30s, 12 cycles of 95C for 10s, 55C for 30s, and 72C for 1min, and one cycle of 72C for 5min. The purified cDNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq2500 following the manufacturer's protocol Less...