Decitabine maintains hematopoietic precursor self-renewal by preventing repression of stem cell genes by a differentiation-inducing stimulus

Abstract

The cytosine analogue decitabine alters hematopoietic differentiation. For example, decitabine treatment increases self-renewal of normal hematopoietic stem cells. The mechanisms underlying decitabine-induced shifts in differentiation are poorly understood, but likely relate to the ability of decitabine to deplete the chromatin-modifying enzyme DNA methyltransferase 1 (DNMT1), which plays a central role in transcription repression. HOXB4 is a transcription factor that promotes hematopoietic stem cell self-renewal. In hematopoietic precursors induced to differentiate by the lineage-specifying transcription factor Pu.1 or by the cytokine granulocyte-colony stimulating factor, there is rapid repression of HOXB4 and other stem cell genes. Depletion of DNMT1 using shRNA or decitabine prevents HOXB4 repression by Pu.1 or granulocyte-colony stimulating factor and maintains hematopoietic precursor self-renewal. In contrast, depletion of DNMT1 by decitabine 6 hours after the differentiation stimulus, that is, after repression of HOXB4 has occurred, augments differentiation. Therefore, DNMT1 is required for the early repression of stem cell genes, which occurs in response to a differentiation stimulus, providing a mechanistic explanation for the observation that decitabine can maintain or increase hematopoietic stem cell self-renewal in the presence of a differentiation stimulus. Using decitabine to deplete DNMT1 after this early repression phase does not impair progressive differentiation.

Figure 1. Dnmt1 depletion by shRNA prevented Pu.1 mediated repression of stem cell associated genes and allowed continued self-renewal in the presence of nuclear Pu.1

PUER are Pu.1 −/− murine hematopoietic precursors retrovirally transduced to express Pu.1 fused to the estrogen receptor. Tamoxifen (OHT) 100nM translocates Pu.1 to the nucleus and triggers macrophage differentiation. A) After addition of OHT, there is repression of stem cell associated genes HoxB4, Bmi-1 and c-Kit, followed by activation of genes associated with macrophage differentiation Mcsfr, Gmcsfr, F4/80. Giemsa-stained cytospin preparations 48h after addition of OHT. Gene expression measured by RQ-PCR, average of 6 independent experiments. B) Dnmt1 was knocked-down in PUER by lentiviral transduction of shRNA (PUER shDnmt1) with PUER transduced with empty vector as control (PUER Control). Dnmt1 measured by Western blot. C) Addition of OHT to shDnmt1 cells caused Pu.1-ER to be rapidly translocated from the cytoplasm into the nucleus at high levels. Pu.1-ER measured by Western blot in nuclear and cytoplasmic fractions. Actin in nuclear fraction. D) OHT treated shDnmt1 cells continued to express high levels of stem cell associated genes and low levels of differentiation genes. Gene expression by RQ-PCR, average of 3 independent experiments. Giemsa-stained cytospin preperations 48h after OHT addition. E) OHT treated shDnmt1 cells proliferated at a lower rate than shDnmt1 cells without OHT, however, the cells continued to exponentially expand at a significantly higher rate than OHT treated Control. Cell counts by automated cell counter, average of 3 independent experiments. Error bars = standard error.

Figure 2. Maintenance of hematopoietic precursor (PUER) self-renewal by decitabine (DAC) 0.5 µM requires timing of decitabine treatment to prevent Pu.1 mediated stem cell gene repression

A) DAC addition concurrent with OHT (Pu.1), but not 6 hours after OHT, attenuated the repression of stem cell genes (HoxB4, Bmi-1 and c-Kit), and the activation of differentiation genes (F4/80, Gmcsfr, and Mcsfr ) compared to OHT alone. Gene expression by RQ-PCR, average of 4 independent experiments. Giemsa-stained cytospin preparations 48h after OHT. B) DAC addition concurrent with OHT (Pu.1) attenuated the decrease in proliferation, and protein expression changes, seen with OHT alone. Cells counts by automated cell counter, average of 3 independent experiments. Error bars = standard error. c-Kit and F4/80 protein expression measured by flow-cytometry.

Figure 3. Cell fate in response to decitabine (DAC) 0.5 µM treatment varies with timing in relationship to G-CSF differentiation stimulus

Human CD34+ hematopoietic precursors were cultured with SCF, TPO, FLT3, IL-3, IL-6 with or without G-CSF and decitabine. A) DAC concurrent with G-CSF, but not 6 hours after G-CSF, significantly attenuated repression of HOXB4, BMI1 and c-KIT, and attenuated activation of PU.1, MCSFR or GCSFR, compared to G-CSF alone. Gene expression by RQ-PCR, average of 4 independent experiments. Giemsa-stained cytospin preparations on day 7 after G-CSF addition. B) DAC addition concurrent with G-CSF decreased the number of progenitors but increased size of colonies formed. DAC 6h after G-CSF decreased both number and size of colonies. Cells harvested from liquid culture 9 days after addition of G-CSF. Cells plated into methyl-cellulose at 20,000 cells per ml of semi-solid media. Colonies counted/photographed Day 12 of semi-solid culture (n=4). C) DAC concurrent with G-CSF decreased cell proliferation compared to G-CSF alone, however, not to the same extent as DAC 6 hours after G-CSF. Cell counts by automated cell counter, average of 4 independent experiments. Error bars = standard error.