Page 67 - Haematologica March 2020

Genetic and epigenetic regulation of latexin transcription
and found that Ets-1 did not bind to Lxn promoter (data not shown). These results prompted us to use the “DNA-pull down” and mass spectrometry to directly look for proteins that bind to this region. We found 15 candidate binding proteins (Table 1). HMGB2 and H2A.X are of particular interest because of the involvement of their family mem- bers in the regulation of stem cell function and differenti- ation as the chromatin modifiers.25-34 We thus focused on these two proteins and examined their role in Lxn tran- scriptional regulation.
We performed ChIP-qPCR assay to measure the binding and occupancy of HMGB2 and H2A.X to the Lxn promot- er. The result showed that HMGB2, but not H2A.X, were significantly enriched at the Lxn promoter region in com- parison to IgG control and to the control region at the downstream 500 bps of the promoter (Figure 1C and D). This result supports the idea of the specific binding of HMGB2 to Lxn promoter. We next performed luciferase assay, and found that HMGB2 suppressed Lxn promoter activity (Figure 1E). Altogether, to our knowledge, this is the first time HMGB2 has been identified as a novel tran- scription suppressor of Lxn gene.
HMGB2 knockdown increases Lxn expression and changes the function of a HSC cell line
We next examined the regulatory role of HMGB2 in Lxn transcription and its effects on EML cells. EML is the only known hematopoietic cell line with both lympho and myelo-erythroid differentiation potential, and is considered to represent HSC.35-37 Knockdown HMGB2 in EML cells significantly increased Lxn expression at both mRNA and protein level (Figure 2A and B, respectively), reinforcing the finding of transcriptional suppression of HMGB2 on Lxn expression. We previously reported Lxn as a negative regulator of HSC number,17 we thus hypothesized that knockdown of HMGB2 would decrease EML cell number via upregulation of Lxn. We therefore monitored the growth of EML cells with HMGB2 knockdown for two weeks, and found that HMGB2 knockdown led to a dramatic decrease in the cell number compared to control group (Figure 2C). Since HMGB2 is a chromatin binding protein, its effect on EML number may not act solely through Lxn upregula- tion. We simultaneously knocked down Lxn in HMGB2 shRNA-transduced cells, and determined whether block- ing Lxn upregulation could attenuate HMGB2-induced growth inhibition. We confirmed that the expression of Lxn mRNA and protein in HMGB2-shRNA transduced EML cells was reduced by the co-transduction with Lxn- shRNA (Figure 2D and E). Figure 2F shows that HMGB2 knockdown significantly decreased EML cell number, and the concomitant Lxn knockdown reversed this change, resulting in an increase in the cell number to the level comparable to control group. These data imply that Lxn is one of the downstream transcriptional targets of HMGB2, and that HMGB2 suppresses Lxn transcription which in turn increases EML number.
We previously reported that Lxn negatively regulates HSC function through increasing apoptosis and decreas- ing proliferation,23,38,39 we hypothesized that HMGB2 inhibition may have similar effects on EML cells. Indeed, we found that knocking down HMGB2 significantly increased the percentage of apoptotic EML cells (Figure 3A). The change was further confirmed by the increased proportion of active caspase 3 positive cells (Figure 3B).
Moreover, knocking down HMGB2 significantly decreased the percentage of cells in the S phase in the cell cycle (Figure 3C). The concomitant increase in apoptosis and decrease in proliferation by HMGB2 inhibition may contribute to the decreased cell number (Figure 2B). We next tested whether apoptosis and proliferation could be rescued by blocking Lxn upregulation in HMGB2 knock- down condition (see also Figure 2E and F). The results showed that Lxn knockdown on the top of HMGB2 knockdown decreased apoptosis (Figure 3D) and the pro- portion of active caspase 3 positive cells (Figure 3E), and restored their changes to the level of control group. However, the cell cycle changes were not fully restored (Figure 3F), suggesting that Lxn may be a major player in regulating apoptosis, and other downstream targets of HMGB2 might be involved in cell cycle regulation that counteract Lxn function. Overall, these data suggest that HMGB2 positively regulates HSC function via the sup- pression of Lxn expression.
HMGB2 knockdown increases Lxn expression in bone marrow hematopoietic stem cells and decreases their number and regenerative function
Because of the observed effect of HMGB2 on Lxn expression and EML function, we next asked whether HMGB2 plays a similar role in primary HSC. We knocked down HMGB2 in bone marrow lineage- Sca-1+ c-Kit+ (LSK) cells (Figure 4A, left), which are enriched with HSC and hematopoietic progenitor cells (HPC), and then determined the effect of HMGB2 knockdown on Lxn expression and HSC and HPC cell numbers, apoptosis and cell cycling. We found that knockdown HMGB2 in LSK cells also led to a significant increase in Lxn expression at both transcript and protein levels (Figure 4A, middle and right). We next performed in vitro short-term CFC and long-term CAFC assays to deter- mine functional HPC and HSC, respectively. The result showed that the numbers of HPC and HSC in HMGB2- knockdown cells were nearly 2-fold lower than those in control cells (Figure 4B and C). Moreover, HMGB2 knockdown led to an increase in apoptosis (Figure 4D) and the proportion of active caspase 3 positive cells (Figure 4E), and a decrease in proliferation in LSK cells (Figure 4F), similar to those seen in the EML cells. These data confirm that HMGB2 also regulates Lxn transcrip- tion in primary HSC, and thereby affects the number and clonality of HSC and HPC.
We next performed a more stringent transplantation experiment to determine the effect of HMGB2 inhibi- tion on HSC regenerative capacity in vivo. Donor cells are LSK cells that were transduced with either HMGB2 shRNA or control shRNA. They were next transplanted into the myeloablated recipient mice with helper cells, and blood and BM regeneration were examined at 16 weeks post transplantation (Figure 5A). The results showed that HMGB2 knockdown resulted in significant decreases in the regeneration of blood cells, BM HSC/HPC-enriched LSK cells, and the most primitive long-term HSC with unlimited self-renewal capacity (Figure 5B-D). These results suggest that HMGB2 inhibi- tion impairs HSC regenerative functionality. Altogether, our data obtained from the EML cell line and primary HSC strongly support the idea that the HMGB2 sup- presses Lxn expression, which in turn affects HSC and HPC number and function.
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