Identification of a Novel Protein-Coding Gene (TIHL) and Its Functional Relevance in Myeloid Cells.

Abstract 2333

The gene locus BX648577 (FLJ27352/hypothetical LOC145788) was recently identified as part of a gene fusion with Class II Transactivator (CIITA) in Hodgkin's lymphoma using whole-transcriptome paired-end sequencing (Steidl C. et al., Nature 2011). While CIITA has been extensively studied, no function is known for BX648577. We therefore sought to study its expression and biological function in normal and malignant hematopoietic cells, including its effects on cellular proliferation, clonogenicity and cell death. Here, we report the endogenous expression of functional mRNA and protein encoded by the BX648577 locus, thereby establishing it as a novel protein-coding gene, which we named TIHL(translocated in Hodgkin's lymphoma).

Initial comparative analysis of the 13–16kDa predicted BX648577 (TIHL) protein sequences in various species revealed high evolutionary conservation (≥54%), including invertebrates (e.g. Saccoglossus kowalevskii). We generated a TIHL(BX648577) -specific antibody and identified endogenous protein expression of TIHL by western blot analysis in several cell lines, including 293T cells, and myeloid NB4 and KG1a cells. Expression analysis was performed in several leukemia and lymphoma cell lines by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). And we found that TIHL mRNA was widely and variably expressed, with particularly high expression in NB4 and THP1 myeloid leukemia cells, 5 fold (p=2.02e-6) and 9 fold (p=6.96e-7) higher, respectively, when compared to healthy donor peripheral blood CD14+ monocytes. In addition, TIHL mRNA expression was detectable in sorted, primary human and murine bone marrow derived hematopoietic stem cells (Lin⁻CD34⁺CD38⁻ (human) or Lin⁻c-Kit⁺Sca-1⁺ (mouse)) and progenitor cells (Lin⁻CD34⁺CD38⁺ (human) or Lin⁻c-Kit⁺Sca-1⁻(mouse)), as well as human healthy donor mature peripheral blood mononuclear cells with the most prominent expression in Glycophorin A and CD56 positive cells by qRT-PCR.

Utilizing primary immature murine bone marrow cells (sorted Lin⁻c-Kit⁺ cells) as well as acute myeloid leukemia cell lines (NB4, KG1a); we assessed the biological consequences of modulating TIHL expression. Knockdown of TIHL by two independent shRNAs with knockdown efficiencies of 77% and 50% respectively, led to significantly decreased leukemic cell growth in suspension culture. We additionally observed decreased clonogenic capacity in both cell lines when the cells were transduced with either sh#1 or sh#2, with 76% (p =1.19e-3) and 77% (p=8.36e-4) inhibition in clonogenicity in NB4 cells when compared to cells transduced with a non-silencing control vector. Similarly, we observed 67% (p=3.28e-3) and 72% (p=4.9e-4) inhibition of clonogenicity in KG1a cells. In addition, knockdown of TIHL led to a 7–14 fold increase in specific apoptosis. We did not find changes indicative of differentiation when we analyzed cellular morphology and surface protein expression. We cloned the full length cDNA of TIHL of human and mouse into a lentiviral expression vector. While ectopic expression of human or murine TIHL in sorted Lin⁻c-Kit⁺cells did not lead to a change in clonogenicity when compared to the empty control vector, we found a 1.5 fold increase in colony forming capacity (p=0.0161), and a 2 fold increase in cell growth (p<0.05) in NB4 cells upon TIHL overexpression.

Finally, in silico domain analysis suggests that TIHL may function as an adaptor protein and may be involved in facilitating previously established signal transduction pathways.

In summary, we here identify TIHL (former gene locus BX648577) as a novel protein-coding gene. We show that TIHL is expressed at the mRNA and protein level with wide expression in both leukemia cell lines and normal hematopoietic cells, including stem cells. We demonstrate that TIHL is functionally relevant and that it is involved in the regulation of growth in myeloid cells. Further studies are warranted to better understand the biological role of TIHL, to examine its precise mechanisms of action, and to delineate the biological pathways implicated in its function.