The actin family member Arp6 and the histone variant H2A.Z are required for spatial positioning of chromatin in chicken cell nuclei

The spatial organization of chromatin in the nucleus contributes to genome function and is altered during the differentiation of normal and tumorigenic cells. Although nuclear actin-related proteins (Arps) have roles in the local alteration of chromatin structure, it is unclear whether they are involved in the spatial positioning of chromatin. In the interphase nucleus of vertebrate cells, gene-dense and gene-poor chromosome territories (CTs) are located in the center and periphery, respectively. We analyzed chicken DT40 cells in which Arp6 had been knocked out conditionally, and showed that the radial distribution of CTs was impaired in these knockout cells. Arp6 is an essential component of the SRCAP chromatin remodeling complex, which deposits the histone variant H2A.Z into chromatin. The redistribution of CTs was also observed in H2A.Z-deficient cells for gene-rich microchromosomes, but to lesser extent for gene-poor macrochromosomes. These results indicate that Arp6 and H2A.Z contribute to the radial distribution of CTs through different mechanisms. Microarray analysis suggested that the localization of chromatin to the nuclear periphery per se is insufficient for the repression of most genes

Gpr177 is dynamically expressed in mammary development.

<p>The expression of Gpr177 in the luminal and basal/myoepithelial cells was analyzed by co-immunostaining with K18 (A–D, M–P), K14 (E–H, Q–T), and SMA (I–L, U–X), respectively. Sections of the virgin 1 month (v1M; A–L) and 2 month (v2M; M–X) mammary gland were co-immunostained with Gpr177 (green) and cell type-specific marker (red) and counterstained with DAPI (blue). Scale bars, 50 µm (A–X).</p

Gpr177 regulates Wnt production and signaling essential for mammary development.

<p>(A) RT-PCR analyzes the Wnt transcripts present in the primary mammary epithelial cells (MEC) isolated from the P21 control and Gpr177^MMTV glands. Immunostaining of Wnt10a (B, C) and activated β-catenin (Act. β-cat, D, E) reveals Wnt signaling, but not Wnt expression, affected by Gpr177 deficiency. (F–I) Wnt production and signaling are analyzed by the use of mouse L (-) and L-Wnt3a (Wnt3a) cell lines. (F) Immunoblot analysis reveals that the secretion of Wnt is prohibited by the knockdown of Gpr177 in the signal-producing cells. (G) Relative luciferase activity (RLA) analysis of the signal-receiving cells, harboring the TOPFLASH reporter, detects activation of the canonical Wnt pathway in the signal-producing cell media. (H) Immunoblot analysis shows the transient expression of the myc tagged Wnt1 (MT-Wnt1) and myc-tagged Gpr177 (MT-Gpr177), as well as the endogenous Gpr177 (Gpr177) in the signal-producing cells, and the level of Act. β-cat in the signal-receiving cells. The level of actin and total β-catenin (β-cat) is used as loading control. (I) RLA analysis of TOPFLASH examines activation of Wnt/β-catenin signaling in the condition media. Scale bars, 50 µm (B–E).</p

The Wnt-mediated proliferation and self-renewal of mammary stem cells (MaSCs) requires Gpr177.

<p>(A) Statistical analysis shows the percentage of mitotic cells positive for immunostaining of Ki67 in the control, MMTV-Wnt1, MMTV-Wnt1; Gpr177^MMTV mammary gland. <i>P</i> values indicate significance of the study (n = 3). (B) Mammosphere analysis examines the self-renewing and proliferation abilities of MaSCs in primary (1⁰) and secondary (2⁰) cultures of control, MMTV-Wnt1 and MMTV-Wnt1; Gpr177^MMTV. The effect of IWP-2 on the Wnt-mediated stem cell self-renewal and proliferation is also investigated. Data represent the mean ± SEM in three independent experiments. Asterisks indicate the <i>p</i> value (1⁰: *, <0.0062, **, <0.0022, ***, <0.0283 and 2⁰: *, <0.0004, **, <0.0024, ***, <0.0076). (C) Diagram illustrates the average size of spheres is not significantly different in the control (131.9±6.9), MMTV-Wnt1 (124.8±3.2), MMTV-Wnt1; Gpr177^MMTV (124.2±4.3), MMTV-Wnt1+IWP-2 (151.5±6.4) cultures. (D) Diagram illustrates the limiting dilution transplantation and calculation of the success ratio showing the frequency of mammary stem cells in MMTV-Wnt1 and MMTV-Wnt1; Gpr177^MMTV mice.</p

Gpr177 deficiency alleviates the abnormalities of mammary cell types caused by aberrant Wnt expression.

<p>(A–U) Sections of the virgin 2 month (v2M) control (A, D, G, J, M, P, S; genotype: Gpr177Fx/+), MMTV-Wnt1 (B, E, H, K, N, Q, T; genotypes: MMTV-Wnt1; Gpr177Fx/+, MMTV-Wnt1; Gpr177Fx/Fx or MMTV-Wnt1; MMTV-Cre; Gpr177Fx/+), and MMTV-Wnt1; Gpr177^MMTV (C, F, I, L, O, R, U) are analyzed by H&E staining (A–C) and immunostaining of Gpr177 (D–F), Wnt1 (G–I), K6 (J–L), K14 (M–O), K18 (P–R) and SMA (S–U). Scale bar, 50 µm (A–U).</p

The loss of Gpr177 impairs mammary cell proliferation and differentiation.

<p>Cells undergoing mitotic division are detected by immunostaining of Ki67 (A, B) and pHH3 (C, D) in the mammary gland at one month. Sections were immunostained with the antibody (brown) and counterstained with Hematoxylin (blue). Arrows indicate cells positive for immunostaining. Immunostaining of K18 (E, F), Cadherin (G, H), K14 (I, J), SMA (K, L) and Gpr177 (M, N) characterizes the effect of the Gpr177 deletion on specification of mammary cell types. Scale bars, 100 µm (A, B); 50 µm (C–N).</p