American Society of Hematology

Figure 4

$Figure 4. AF10 regulates H3K79 methylation by targeting hDOT1L to chromatin, but fusion to CALM reduces this targeting dramatically. (A) Confirmation of H3K79 hypomethylation in AF10 and hDOT1L knockdown T-REx-293 cells. siRNA expression was induced in the stable cell lines with doxycycline (Dox) for 48 hours (+), and the H3-K79 methylation levels were examined (top panel). The knockdown efficiency was confirmed by reverse transcription-PCR, and ACTIN was used as normalization control (3 bottom panels). (B) Expression of AF10 targeted the cotransfected hDOT1L to chromatin, but CALM-AF10 targeted only very little hDOT1L to chromatin. The 293T cells were cotransfected with expression constructs for hDOT1L-HA and Flag-tagged proteins indicated on the top and subjected to cell fractionation. The distribution of hDOT1L-HA in each fraction was examined by Western analysis with HA antibody (top panel). The efficiency of the cell fractionation was confirmed by the detection of proliferating cell nuclear antigen in the soluble fraction, histone H3 in chromatin, and lamin B in the matrix. (C) Expression of AF10, but not CALM-AF10, targeted cotransfected hDOT1L to chromosomes. Metaphase chromosome spreads were prepared from cotransfected 293T cells. DAPI (4,6 diamidino-2-phenylindole) was used as DNA counterstain. Scale bar indicates 1 μm.$

AF10 regulates H3K79 methylation by targeting hDOT1L to chromatin, but fusion to CALM reduces this targeting dramatically. (A) Confirmation of H3K79 hypomethylation in AF10 and hDOT1L knockdown T-REx-293 cells. siRNA expression was induced in the stable cell lines with doxycycline (Dox) for 48 hours (+), and the H3-K79 methylation levels were examined (top panel). The knockdown efficiency was confirmed by reverse transcription-PCR, and ACTIN was used as normalization control (3 bottom panels). (B) Expression of AF10 targeted the cotransfected hDOT1L to chromatin, but CALM-AF10 targeted only very little hDOT1L to chromatin. The 293T cells were cotransfected with expression constructs for hDOT1L-HA and Flag-tagged proteins indicated on the top and subjected to cell fractionation. The distribution of hDOT1L-HA in each fraction was examined by Western analysis with HA antibody (top panel). The efficiency of the cell fractionation was confirmed by the detection of proliferating cell nuclear antigen in the soluble fraction, histone H3 in chromatin, and lamin B in the matrix. (C) Expression of AF10, but not CALM-AF10, targeted cotransfected hDOT1L to chromosomes. Metaphase chromosome spreads were prepared from cotransfected 293T cells. DAPI (4,6 diamidino-2-phenylindole) was used as DNA counterstain. Scale bar indicates 1 μm.

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