The TRPM7 Chanzyme Is Cleaved to Release a Chromatin-Modifying Kinase

TRPM7 Cleavage Fragments Identified in Multiple Cell Lines and Tissues

(A) TRPM7 protein cleavage fragments in mouse mesangial SV40 mes13 cells. Cells were extracted with Tris-buffered saline (TBS)/1% NP40. Endogenous TRPM7 was immunoprecipitated (IP’d) from lysates with TRPM7 C-terminal mouse monoclonal antibody (αA25) or normal mouse immunoglobulin G (IgG) and probed on western blots (WBs) with anti-C-terminal rabbit antibody (αC47). C-terminally HA-tagged TRPM7 (expressed = expr) was IP’d with anti-HA-agarose (αHA) from SV40 mes13 cells stably expressing recombinant protein and probed on WBs with αHA-peroxidase conjugate. Scale (left) indicates the molecular weight of major bands calculated from their electrophoretic mobility relative to standard molecular weight markers. Cartoon (right) shows the approximate position of cleavage sites; K indicates kinase domain.

(B) TRPM7 cleavage pattern in eight distinct cell lines. Mouse mesangial (SV40 mes13), macrophage (RAW 264.7), mESC, human B-lymphocyte (Raji), Caco-2 (colon epithelial), prostate (metastatic LNCaP and nonmetastatic RWPE1), and human embryonic kidney 293 (HEK293) cells were extracted and IP’d as described in (A). Extracts demonstrate the relative amounts of cleaved TRPM7 isolated from each tissue. Information about the relative content of the full-length TRPM7 and the cleaved fragments is contained in each individual lane, which is intentionally not normalized to control protein. No positive bands were found from the same tissue extracts IP’d with normal mouse IgG (data not shown). mESCs were generated as described in Experimental Procedures from WT or TrpM7^−/− (KO) blastocysts. The lower panel in the mESC column shows equal actin content in both mESC lysates. Samples run on different gels are combined in the figure and aligned against identical molecular weight markers.

(C) TRPM7 cleavage pattern in different mouse tissues. Freshly isolated mouse organs were extracted and IP’d as described in (A). Extracts demonstrate the relative amounts of cleaved TRPM7 isolated from in each tissue. No positive bands were found from the same tissue extracts IP’d with normal mouse IgG (data not shown).

See also Figures S1, S2, and S4.

TRPM7 Binds Transcription Factors and Subunits of Chromatin-Remodeling Complexes

(A) Endogenous TRPM7 was IP’d from combined cytosolic plus nuclear extracts of WT or KO9 mESCs with αA25 mouse or αC47 rabbit antibody and the IP probed on WBs with antibody to the indicated proteins. INO80 cell lysate lanes were run on a different gel, as indicated by the gap.

(B) Subunits of Polycomb and INO80 chromatin-remodeling complexes bind ectopically expressed M7CKs. FLAG-tagged transcription factors were transiently coexpressed in 293T cells with HA-tagged M7CK-L, M7CK-M, or M7CK-S. Cytosolic plus nuclear extract was IP’d with αHA-agarose, and co-IP’d proteins were probed on WBs with αFLAG-peroxidase.

(C) The affinity-purified long form of M7CK (M7CK-L) binds purified transcription factors (Coomassie). Left three panels: HA-tagged M7CK-L (M7) was combined with purified YY1 or Ezh2 protein complexes and IP’d with αHA-agarose. Right: purified GST or GST-RYBP fragment (M7CK-binding domain, amino acids 22–47; GST-RYBP_bd) fusion proteins were combined with purified M7CK-L and GST pulled down with glutathione agarose.

See also Figures S2 and S3.

Nuclear Location of M7CK

(A) TRPM7 cleavage fragments (M7CK) in the nucleus and cytosol. HA-tagged TRPM7 C-terminal fragment (amino acids 1,299–1,864; green) expressed transiently (HEK) or stably (SV40 mes13) immunostained with αHA. Nuclei were stained with propidium iodide (red).

(B) Bound nuclear proteins enable M7CK nuclear accumulation. HEK cells were cotransfected with the indicated HA-tagged M7CK and FLAG-tagged nuclear protein or empty vector. Relative nuclear and cytosolic M7CK concentration was measured as average fluorescence intensity in confocal images of formaldehyde-fixed cells immunostained with epitope-tagged antibodies; 60–70 cells were measured for each condition. Cumulative frequency plots demonstrate that coexpression with the bound nuclear protein increases the fraction of the cells with higher nuclear M7CK concentration. p, Kolmogorov-Smirnov probability.

Zinc Dependence of M7CK Binding to Zinc-Finger Domain-Containing Proteins

(A) HA-tagged M7CK was coexpressed with FLAG-tagged interacting proteins in 293T cells. The cell lysate was split into equal aliquots and supplemented with 20 μM TPEN and 50 μM of the indicated ions. M7CK was IP’d with αHA and the co-IP’d proteins probed with αFLAG.

(B) FLAG-tagged zinc-finger domain (ZfD)-containing proteins were coexpressed in 293T cells with HA-tagged M7CK. [Zn²⁺] was adjusted by variation of [EGTA] and [ZnSO₄]. M7CK-HA was IP’d with HA-agarose; ZfD protein binding was detected in the αFLAG WB.

(C) [Zn²⁺] dependence of M7CK binding to GST-RYBP ZfD. Purified GST fusion with RYBP fragment (amino acids 22–47 comprising the RYBP-ZfD) was combined with purified M7CK-L in the presence of varying [Zn²⁺] and pulled down by glutathione agarose (Coomassie-stained gel).

(D) Quantification of M7CK binding shown in (C). Values of integrated pixels of the bound M7CK image were normalized to maximum binding at 10 μM [Zn²⁺]. Fitted equation and parameters shown.

See also Figure S4.

Cytosolic [Zn²⁺] Measured in WT and TrpM7^−/− mESCs

(A) Fluorescent image of mESCs stably expressing the Zn²⁺ indicator, eCALWY-4.

(B) eCALWY-4 fluorescence recordings from multiple cells in a single sample.

(C) Calculated cytosolic [Zn²⁺] shown as mean ± SEM.

(D) Kolmogorov-Smirnov statistics.

(E) Histogram of [Zn²⁺] in cell populations for WT3 and TrpM7^−/− (KO9) mESC 260 cells of each type were measured in four independent experiments.

See also Figure S5.

TRPM7 Kinase-Dependent Global Histone Phosphorylation and Acetylation

Bar graphs display histone modification data statistics (mean ± SD, normalized to J1 data) for three independent experiments.

(A) Global H3S10, H3S28, and H3T3 phosphorylation is decreased in TrpM7^−/− cells. WBs of mESC histone extracts with specific anti-phosphoserine antibodies.

(B) Restoration of histone H3S10 and H3S28 phosphorylation to WT levels by stable expression of active M7CK-L in TrpM7^−/− mESCs (KO9+CKa). Kinase-dead M7CK mutant (KO9+CKi) expression further decreases H3S10 and H3S28 phosphorylation.

(C) Decrease in global H3 histone Lys9 and Lys27 acetylation (H3K9Ac and H3K27Ac) in Trpm7^−/−mESCs as shown by WB with anti-acetylated lysine antibodies.

(D and E) WBs and summary of global H2AX phosphorylation in mESCs.

(F) Attenuation of γH2AX to WT levels by stable expression of active M7CK-L in TrpM7^−/− mESCs. Expression of kinase-dead M7CK mutant further increases H2AX phosphorylation.

(G–I) TRPM7 IP’d from mESCs pulls down H3, but not H2AX (G). Purified M7CK directly phosphorylated purified histones as shown by incorporation of radioactive phosphate from [γ-³³P]-ATP (H) and WBs with specific phosphoserine antibody (I).

See also Figure S6.

M7CK-Dependent H3S10 Histone Phosphorylation in Gene Promoters Correlates with Gene Expression

(A) M7CK-L expression in TrpM7^−/− mESC rescues activity of downregulated genes. Expression of selected genes was quantified with quantitative RT-PCR (mean ± SD) in WT3, TrpM7^−/− (KO9), and KO9 stably expressing M7CK-L (KO9+CKa) mESCs.

(B) H3S10 phosphorylation in promoters of genes affected by TRPM7 deletion in mESC was attenuated in TrpM7^−/− (KO9) cells and restored by M7CK expression (KO9+CKa), as determined by ChIP-qPCR (mean ± SD).

Cleavage Properties of Endogenous TRPM7, Related to Figure 1

(A) Endogenous TRPM7 cleavage is independent of time after cell lysis. SV40 mes13 cells were lysed in cold buffer containing protease inhibitor cocktail. The cleared supernatant was IP’d with αA25 antibody for the time indicated and probed on WB with αC47 antibody. Cell lysis and centrifugation ∼40 min.

(B) TRPM7’s cleavage pattern changes with embryonic stem cell differentiation into embryoid bodies. IP and WB were performed as described in the legend to Figure 1. Left lane (KO): TRPM7 in 12 d embryoid bodies generated from TrpM7^−/− mESC. Numbers at top indicate days of differentiation.

(C) M7CK cleavage fragments were not detected in mESC neuronal cells. Wild-type (WT3 clone) and TrpM7^−/− (KO9 clone) were differentiated into neural precursors in monolayer culture as described (Ying et al., 2003) with an additional 10 mM MgCl₂ in the media in order to maintain proliferation of KO9 cells. On day 14 of differentiation, cells were lysed and TRPM7 was detected as described in Figure 1. Image (left) is Tubulin-βIII (green) immunofluorescence of neural precursors on the 16^th day of differentiation (propidium iodide stains nuclei red). Note: In contrast to TrpM7^−/− mESC, neural precursors differentiated from TRPM7^−/− mESC cells do not require elevated [Mg²⁺] to maintain proliferation. Also, H3S10 and H3S28 phosphorylation did not differ between neural precursors derived from WT3 and KO9 mESC. This result is consistent with the lack of M7CK in neural precursors and the proposed role of M7CK in H3 histone phosphorylation.

(D) The endogenous TRPM7 channel moiety is not detected on western blot after kinase cleavage. Stop codons were introduced into N-terminal HA-tagged TRPM7 cDNA to produce TRPM7 truncated after amino acids 1444 (Δ1445), 1396 (Δ1397) and 1298 (Δ1299), corresponding to truncations produced by kinase domain cleavage sites (see Figure S3A). Upper panel: TRPM7 mutant constructs were transiently expressed in 293T cells, IP’d and western-blotted with αHA antibody. Lower panel: TRPM7 was IP’d with αNFP antibody (recognizing the epitope on the TRPM7 N terminus) from cells with high levels of TRPM7 cleavage (>90% in mouse kidney, 50%–70% in SV40 mes13 cells) and western-blotted with αCFP antibody recognizing epitope located at the TRPM7 C terminus. 1 μg SV40 mes13 and 6 μg mouse kidney lysate were used for IP. To increase band resolution, proteins were separated on 3%–8% Tris-acetate gel (Invitrogen). Note that αCFP antibody did not recognize the Δ1299 protein.

(E) TRPM7 mRNA was equally expressed (left panel; qPCR data, mean ± SD) in normal (J1) and the Δkinase (TRPM7 truncated after amino acid 1537; Ryazanova et al., 2010) mESC, but truncated TRPM7 protein (right panel shows western blot, TRPM7 αCFP antibody) was not detected in TRPM7-Δkinase mESC. Arrow indicates predicted position for the TRPM7-1537-STOP protein.

TrpM7^−/− Mouse Embryonic Stem Cells, Related to Figures 1 and 2 and Experimental Procedures

Blastocysts obtained from mating of TrpM7^+/− mice (Jin et al., 2008) were collected and expanded in tissue culture to establish ES cell lines using standard procedures. Both WT and TRPM7^−/− mESC were cultured in media containing an additional 10 mM MgCl₂. For these assays, cells were passaged 3 times without feeder cells.

(A) Genotyping of clonal mESC as described in (Jin et al., 2008).

(B) RT-qPCR quantification of TRPM7 RNA in J1 (ATCC SCRC-1010), WT3, TrpM7^−/− clones 9 and 12 (KO9, KO12) and TRPM7^+/− clone 11 (HZ11) mESC. The minor content of TRPM7 RNA in KO cells likely originated from traces of feeder cells. Absence of TRPM7 protein in KO cells is shown in Figure 1.

(C–E) TrpM7^−/− mESC form normal colonies when grown on feeder cells (C) and differentiate into embryoid bodies morphologically indistinguishable from WT (D) but proliferate only with an additional 10 mM MgCl₂ in the media (E). Cell proliferation was quantified using an MTT assay. Note that additional Mg²⁺ did not affect proliferation of WT cells.

(F) RT-qPCR quantification (mean ± SD) of stem cell markers in clonal mESC. CKa = kinase active, CKi = kinase dead. As for parental KO9 mESC, M7CK-expressing cells proliferate only in media containing an additional 10 mM Mg²⁺ (data not shown).

Binding Properties of M7CKs, Related to Figure 2

(A) Comparison of the electrophoretic mobility of endogenous (endo; SV40 mes13) with ectopically expressed (HEK293T) untagged TRPM7 C-terminal cleaved fragments. TRPM7 fragments contained amino acids 1299-1864 (L)ong), 1397-1864 (M)edium) and 1445-1864 (S)hort). M7CKs were IP’d and western blotted as described in the legend to Figure 1.

(B) Endogenous DDB1 binds TRPM7 in mESC. Endogenous TRPM7 was IP’d from WT3 or TrpM7^−/− (KO9) mESC and DDB1 was detected on WB with αDDB1 antibody.

(C) Cul4B binds active (CKa) and kinase-dead mutant (CKi) M7CK-L when coexpressed in HEK293 cells.

(D and E) DBC1 and Sirt1 bind M7CKs when coexpressed in HEK293 cells.

(F) Recombinant M7CK-L HA-tagged protein expressed in E. coli and affinity purified. Figure displays Coomassie-stained gel (protein) and western blots with HA-tag antibody (αHA) and antibody to the C terminus of TRPM7 (αC47) of purified M7CK-L protein.

(G) TrpM7^−/− mESC stably expressing active (CKa) and the kinase-dead mutant (K1646A mutation, CKi) C-terminally HA-tagged M7CK-L. Clones expressing comparable levels of active (KO9+CKa) and inactive (KO9+CKi) kinase were selected for further experiments. ³³P designates radioactive phosphate incorporated into M7CK in the autophosphorylation assay.

(H) Kinase activity regulates protein binding to M7CK. Active (A) and kinase-dead (K1646A mutation, I) HA-tagged M7CK-L was co-expressed with FLAG-tagged interacting proteins in 293T cells. M7CK was IP’d from the cell lysate with αHA and co-IP’d proteins were probed with αFLAG.

WT and Dominant-Negative Pore Mutant TRPM7 Are Identically Cleaved, Related to Figures 1 and 4

HEK293 and SV40 mes13 cells were transiently transfected with a TRPM7 cDNA construct containing an HA epitope tag on the C terminus. TRPM7 was IP’d with αHA-agarose and stained on western blot with αHA-peroxidase conjugate. PM, pore mutant.

Cytosolic [Mg²⁺] Is Equal in WT and TRPM7^−/− mESC, Related to Figure 5

Feeder-free cells were passaged x 3 in standard media (J1) or in media supplemented with 10 mM MgCl₂. 2 hr before measurement, the media was replaced with either the same media or, where indicated, with media containing normal [Mg²⁺]. Measurements were in mESC Ringer solution ± 10 mM MgCl₂. Data are mean ± SD; ∼120 cells in each experiment.

Loss of TRPM7 Does Not Affect Global DNA Damage or the Cell Cycle, Related to Figure 6

(A) TrpM7 gene deletion does not increase double-strand DNA breaks in mESC. Double-strand DNA breaks are revealed by foci (arrow) formed by activated ATM kinase. Images depict immunofluorescent staining with antibody recognizing active ATM (phosphorylated Serine 1981, green) and nuclei staining with propidium iodide (red). Irradiated mouse embryonic fibroblasts (mEF-IR) serve as a positive control.

(B) The percentage of mitotic cells, as detected by phosphorylation of H3S10 and H3S28, is not decreased in TrpM7^−/− (KO9) mESC. mESC were labeled with H3S10p and H3S28p anti-phosphoserine antibodies (green). Nuclear DNA was labeled with propidium iodide (red). A total of 1,200 cells were analyzed in 3 independent samples for each mESC clone.