Sara Þöll Halldórsdóttir and Hans Tómas Björnsson
Introduction: We have preciously observed that two disease relevant cell types in Kabuki syndrome (KS), KMT2D-deficient neurons and chondrocytes show precocious differentiation resulting in premature maturation. Growth retardation is a defining characteristic in KS and is seen in majority of individuals but KMT2D involvement in chondrocyte differentiation remains unclear. Prior work has also demonstrated elevated levels of HIF1A in KMT2D-deficient neurons and hypoxia is known to be a vital physiological factor in chondrocyte differentiation. We hypothesized that KMT2D deficiency increases Hif1a transcription which plays a role in driving cells towards chondrocyte differentiation.
Methods: Murine chondrocyte cell line ATDC5 was differentiated in normoxic (20% O2) and hypoxic (1% O2) conditions. Differentiation rate was quantified with Alcian Blue staining. scRNA sequencing was performed over four differentiation time-points, RNA sequencing on undifferentiated cells both in normoxia and hypoxia and CUT&RUN interrogating H3K4me1, H3K4me3 and KMT2D.
Results: scRNA sequencing revealed that KMT2D-deficient cells (Kmt2d-/-) cells are primed towards differentiation even at undifferentiated state. Kmt2d-/- cells exhibit precocious differentiation in normoxia compared to WT cells (p<0.01) but fail to transition to hypertrophic state under hypoxia, resulting in no difference from WT cells (ns). Undifferentiated Kmt2d-/- cells display elevated expression of Hif1a (p<0.01) both in normoxia and hypoxia along with an increase in upregulated HIF1A-targets. However, ncreased transcription of Hif1a in Kmt2d-/- cells is not caused by changes in histone methylation or direct binding of KMT2D suggesting an indirect role. Conclusions: Our results suggest abnormal hypoxia responses in KMT2D-deficient cells that could mediate precocious differentiation.
