Fig. 5: TTC7A is required for the proper histones distribution and chromatin packaging. | Cell Discovery

Fig. 5: TTC7A is required for the proper histones distribution and chromatin packaging.

From: Tetratricopeptide repeat domain 7A is a nuclear factor that modulates transcription and chromatin structure

Fig. 5

a and b Histones subcellular fractionation in control and patient B lymphoblastoid cell lines. Histones H2B, H3, H4, and H1 were analyzed by western blot. Fractions are total histones, cytosolic histones (soluble pool), nuclear histones (salt-extractable pool), and chromatin histones (DNA enwrapped pool). The four controls are compared with six patient samples, and quantification is relative to control that is set to 1. The signal intensities of histones were normalized to internal loading control, α-tubulin (αtub), for total and cytosolic fractions; β-actin (βact) for nuclear and chromatin fractions. Graphs represent the ratio between nuclear vs. cytosolic (a) and chromatin-bound vs. total pools (b). Mean ± SEM of four independent experiments. Unpaired t test; *p-value < 0.05, ***p-value = 0.0003, ****p-value < 0.0001. c DNA lengths distribution upon micrococcal nuclease digestion of chromatin. Lengths of DNA fragments were measured and quantified using the fragment analyzer software. The scheme depicts the range of DNA sizes forming to subnucleosomal DNA (centered around 60 bp), forming the nucleosomal core particle (centered around 145 bp) and forming the chromatosome (centered around 165 bp). LM: lower marker, UM: upper marker. d A kinetic of micrococcal nuclease digestion of chromatin was conducted in control and patient B lymphoblastoid cell lines (P1_E71K, P2_E71K, and P */L478P). DNA concentration was measured and normalized to total DNA concentration. The mean of each time point was compared between control and patients. Mean ± SEM of six independent experiments. Sidak’s multiple comparisons test; *p-value < 0.05, ***p-value < 0.001, ****p-value < 0.0001. e Micrococcal nuclease digestion kinetics same as (d). Control and patient cells lines were stably expressing Flag-tagged WT_TTC7A. The mean of each time point was compared between control and patients. Mean ±  SEM of five independent experiments. Sidak’s multiple comparisons test; ns non-significant

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