HAT2 mediates histone H4K4 acetylation and affects micrococcal nuclease sensitivity of chromatin in Leishmania donovani

Abstract

Histone post-translational modifications (PTMs) such as acetylation and methylation are known to affect chromatin higher order structures. Primary targets of these modifications include basic residues present at N-terminus tail region of core histones. Four histone acetyltransferase (HAT) genes have been identified in trypanosomatids. HAT1, HAT3 and HAT4 of Leishmania donovani have been partially characterized. However, there is no report about HAT2 of Leishmania donovani. Lysine residues present on the N-terminal tail of Leishmania donovani histone H4 are conserved in other trypanosomatids and humans. PTMs of lysines modulate various functions at chromatin level. The four histone acetyltransferases encoded in Leishmania genome were over-expressed to analyse their functional activity. All four HATs were found actively acetylating core histones H3/H4. Similar to L. donovani HAT3 and HAT4, HAT2 was found to be a member of MYST family protein and have SAS2 type domain. Over-expression of HAT2 significantly increases acetylation of H4K4. To analyse the effect of HAT2 over-expression on chromatin accessibility, micrococcal nuclease digestion assay was performed. MNase digestion resulted in a higher proportion of the mononucleosomes and dinucleosomes in HAT2 over-expressing cells as compared to WT L. donovani cells. Acetylation of lysine-4 neutralizes the amino terminal region of histone H4. This weakens its interaction with neighbouring nucleosomes and the linker DNA. HAT2 over-expression in L. donovani resulted in highly accessible chromatin suggesting chromatin decondensation. HAT2 may have an important role to play in global regulation of transcription in L. donovani. Better understanding of these epigenetic determinants of parasite would help in designing novel therapeutic strategies.

Fig 1. Sequence analysis of Histone H4 and HAT2 of L. donovani.

(1A) Comparison of N-terminus region of histone H4 of L. donovani (LdH4) with that of L. infantum (LiH4), L. major (LmH4), L. mexicana (LmxH4), Trypanosoma brucei (TbH4), T. cruzi (TcH4) and human (HuH4). Boxes represent conserved lysine residues. The arrow represents K16 of human histone H4 which is replaced by arginine (R14) in L. donovani. (1B) Zinc finger motif present in the sequence of HAT2 formed by two cysteine and two histidine residues (C₂H₂ type).

Fig 2. HAT2 over-expression and activity analysis.

(2A) RE double digestion of HAT2 cloned in pLPneo2. Clone was digested with HindIII and XhoI. Two fragments (~1800 base pairs for HAT2 and ~5500 base pairs for vector pLPneo2) were resolved in 1% agarose gel. (2B) WT and HAT2 over-expressing L. donovani cells were counted every eight hours for seven days and plotted. P <0.0001 was observed in two-way analysis of variance showing statistical significance. (2C) Relative histone acetylation activity assay for L. donovani cells over-expressing HAT1, HAT2, HAT3 and HAT4. The activities were compared to WT cells and vector (pLPneo2) alone transfected cells. Nuclear extract supplied with kit was taken as experimental control. Total histone acetylation was measured at 2 hours 30 minutes and 3 hours after start of reaction and relative activities were calculated per minute and per gram of total cell extract. The data shown represent average of three independent experiments. The p values < 0.0001 compared with control values is shown as ***.

Fig 3. Acetylation of Histones H3 and H4 in over-expressing Leishmania donovani.

(3A) Western blot analysis of HAT2 over-expressing L. donovani (Lane 3) using Anti-acetyl-Histone H4 (Millipore, Cat. 06–866) antibody. WT (Lane 1) and vector (pLPneo2) transfected L. donovani (Lane 2) were used as controls. M indicates molecular weight marker. For nuclear loading control, blot was reprobed with anti H3 antibody. (3B) H4K4 acetylation assay using histones isolated from HATs over-expressing cells by colorimetric method. Histones of WT cells and pLPneo2 transfected cells were used as control. Relative levels of acetylation of H4K4 are shown in percentage assuming that of WT cells as 100%. The p values < 0.0001 compared with WT cells are shown as ***. (3C) Total histone H3 acetylation assay by fluorimetric method for HATs over-expressing cells. Histones isolated from WT cells and vector (pLPneo2) only transfected cell were taken as controls. H3 acetylation was presented in percentage considering 100% for WT cells. *** represents p values < 0.0001 compared to WT cells.

Fig 4. MNase digestion of WT and HAT2 over-expressing Leishmania donovani chromatin.

(4A) L. donovani cells (WT and HAT2 over-expressing) were permeabilized with Triton X-100 and treated with varying units of MNase (0.75, 1.5 and 3.0 U/ml) and chromatin was isolated and run on 3% agarose gel. Untreated permeabilized WT and HAT2 overexpressed cells were processed identically (Lane 1 and Lane 6 respectively). In Lane M, molecular weight marker (100 bp ladder) was run. Positions of mononucleosomes and dinucleosomes were indicated for comparing their abundances. (4B) Intensities of bands corresponding to mononucleosomes were quantified using Gene Tools software (Syngene). Statistical significance was checked by two-way analysis of variance followed by Bonferroni post test. ** and *** represents p values < 0.01 and < 0.001, respectively compared to WT cells. (4C) Comparison of dinucleosomal band intensities in WT and HAT2 over-expressing L. donovani cells at varying MNase concentrations (0.75, 1.5 and 3.0 U/ml). Two-way analysis of variance followed by Bonferroni post test was applied to check the statistical significance of differences of band intensities between WT and HAT2 over-expressing cells. P values <0.05 and <0.001 is represented by * and ***, respectively.