Hypomethylation of the promoter region drives ectopic expression of TMEM244 in Sézary cells

Abstract Sézary syndrome (SS) is an aggressive form of cutaneous T‐cell lymphoma (CTCL) characterized by the presence of circulating malignant CD4+ T cells (Sézary cells) with many complex changes in the genome, transcriptome and epigenome. Epigenetic dysregulation seems to have an important role in the development and progression of SS as it was shown that SS cells are characterized by widespread changes in DNA methylation. In this study, we show that the transmembrane protein coding gene TMEM244 is ectopically expressed in all SS patients and SS‐derived cell lines and, to a lower extent, in mycosis fungoides and in a fraction of T‐cell lymphomas, but not in B‐cell malignancies and mononuclear cells of healthy individuals. We show that in patient samples and in the T‐cell lines TMEM244 expression is negatively correlated with the methylation level of its promoter. Furthermore, we demonstrate that TMEM244 expression can be activated in vitro by the CRISPR‐dCas9–induced specific demethylation of TMEM244 promoter region. Since both, TMEM244 expression and its promoter demethylation, are not detected in normal lymphoid cells, they can be potentially used as markers in Sézary syndrome and some other T‐cell lymphomas.

TA B L E 1 TMEM244 expression and promoter methylation in haematological patients, healthy individuals and T-cell lines  9 In cancer, disruption in methylation pattern, especially global hypomethylation of the genome, leads to chromosomal instability and consequently to altered transcription and impaired signalling pathways. 10 In our previous study, we identified ectopic expression of transmembrane protein gene (TMEM244), with unknown biological function, in SS patients but not in healthy individuals. 11 The purpose of this study was to unravel the mechanism responsible for TMEM244 activation. We found a negative correlation between TMEM244 expression and promoter methylation in patient samples and in T-cell lines suggesting methylation to be a mechanism responsible for regulation of TMEM244 expression. This concept was proved in vitro using CRISPR-dCas9 epigenome editing system, by activating TMEM244 expression in Jurkat cells upon specific demethylation of selected CpGs in TMEM244 promoter region.

| Real-time quantitative PCR (RT-qPCR)
RT-qPCR was performed using the CFX96 Real-Time System (Bio-

| Western Blot
Cells were treated with RIPA lysis buffer (Sigma included in the gel, react with tryptophan residues and after activation by UV light produce fluorescent signal that can be quantified in order to measure the relative amount of sample total protein.

| DNA methylation analysis by bisulfite pyrosequencing
The pyrosequencing assay for the analysis of DNA methylation level in TMEM244 promoter region was designed using

| TMEM44 is expressed in SS and CTCL cell lines with hypomethylation of the promoter region
Our previous study showed that TMEM244 is expressed in SS patients (P1-P3 mean ± SD = 681 ± 413E-6). 11 In this study, TMEM244

expression was quantified in different T-and B-cell lymphoma and leukaemia patients, in mononuclear cells of healthy individuals and
in four T-cell lymphoma (TCL) and one TALL cell lines (   and 56% respectively). Three samples from a previous study (P1-

P3) consisted of a heterogeneous population of mononuclear cells.
Therefore, despite 33.5%-67.9% methylation, most likely derived from the admixture of non-malignant cells, the samples showed high TMEM244 expression. In 2/3 MF patients, partial demethylation of TMEM244 promoter in the blood samples (59% and 70%) was accompanied by a moderate TMEM244 expression (198E-6 and 351E-6). In one PTCL and one T-ALL samples with a weak TMEM244 expression, the mean methylation level was only slightly decreased compared with controls (73% and 80% respectively).
Among the 39 samples collected from patients, healthy donors and cell lines, a meaningful TMEM244 expression was observed in 13 samples: five SS, two peripheral blood of MF, one CLL, one T-ALL, one T-cell lymphoma, and three T-cell lines. In those samples, the mean promoter methylation level was 44.11%±30.4 and the mean TMEM244 expression was 1,246E-6 ± 2,206E-6. In the samples with trace or no TMEM244 expression (27E-6 ± 26E-6), the mean methylation level was markedly higher (85% ± 8.5). Based on the obtained results, a cut-off value for promoter hypomethylation was set at 70% and for TMEM244 expression at 100E-6. Using these cut-offs, 11/12 samples with promoter hypomethylation expressed TMEM244 and 25/27 samples with methylated promoter did not express TMEM244 (P < 0.000001 in Fisher exact test; Table 1). Pearson correlation coefficient test showed a strong negative correlation between TMEM244 expression and the square of its promoter methylation ( Figure 2; R = −0.7813; P < .00001).

| In vitro demethylation of TMEM244 promoter activates TMEM244 expression
In order to prove the mechanism of TMEM244 transcriptional activation by promoter demethylation, the CRISPR-dCas9-TET1 system was used for directed demethylation. In the first step, Jurkat cell line was transduced with the dCas9-Tet1 expressing vector. After selection, the expression of this fusion was confirmed on protein level using Western blot and anti-dCas9 antibody ( Figure 3A). Secondly, To check the correlation between TMEM244 expression and promoter methylation, we performed the Pearson correlation coefficient test (Figure 4) for all dCas9-TET1 samples. The analysis showed that the level of TMEM244 expression is negatively correlated with the square of the methylation level in the promoter region (R = −0.4766), and this correlation is highly significant (P < 0.0002) (Figure 4).

| D ISCUSS I ON
In this study, we showed that methylation is a key regulatory mechanism of TMEM244 expression. Samples with TMEM244 expression, among them mostly SS and a few other T-cell leukaemia/ lymphoma cases, had promoter region hypomethylated, while in all samples not expressing the gene, the promoter was methylated.
The negative correlation between TMEM244 expression and promoter methylation was confirmed, and the mechanism was verified using CRISPR-dCas9-TET1 system for directed demethylation of the specific sites in the promoter region. This approach has not been used in CTCL studies so far. The only methylation modification was performed using 5-aza-2'deoxycitidine, a pan-demethylating compound. Upon 5-aza treatment, a down-regulated expression of two tumour suppressors, THBS4 and PTPRG were restored, 9 as well as a potential epigenetic diagnostic marker CMTM2 8 and miR200c involved in activation of Notch pathways in CTCL. 17 The advantage of using CRISPR-dCas9-TET1 approach is its specificity to the region of interest, without affecting global methylation patterns.  Genome-wide methylation analysis in CTCLs revealed that more CpG sites were hypomethylated than hypermethylated. 8,18 Hypomethylation leads to chromosomal instability and is often observed in cancer genomes. However, there are only two reports that actually describe a hypomethylation of specific genes in CTCLs.

Wong et al described hypomethylation-mediated overexpression
of PLS3, GATA3 and TWIST3. 18 GATA3 overexpression in CTCL was confirmed by Kamijo et al 19 The study showed that hypomethylation-mediated GATA6 overexpression promotes tumour progression via overexpression of CD137L that together with CD137 activates pathways leading to cell proliferation, tumour survival, growth and migration.
More studies were published on hypermethylated genes in CTCL, as they are often tumour suppressor genes involved in DNA repair, cell cycle, proliferation and apoptotic pathways. Hypermethylation in the promoter region, followed by decreased expression level, was detected for several tumour suppressors, including CDKN2B (p15), CDKN2A (p16) and MGMT, 20 BCL7A, PTPRG and TP73 (p73) 9 and RUNX3/p46. 21 Promoter methylation not always resulted in gene silencing, and overexpression of IL-15 in CTCL was actually associated with hypermethylation of the promoter, preventing binding of ZEB1 transcription repressor. 22 Little is known about the TMEM244 gene itself. It belongs to a family of transmembrane proteins (TMEMs) that are components of various membranes (cell membranes, mitochondrial, ER, lysosomal, Golgi membranes), present in different cells and fulfil important physiological functions. Many TMEMs are differentially expressed in different cancers. 23 So far, the role of TMEM244 is unknown and no studies have been conducted in order to unravel its function.
Although many RNAseq analysis has been performed for CTCLs samples, 4,24,25 only our team paid attention to that gene, probably due to its relatively low expression.
Our current results show that the expression of TMEM244 gene is associated with T-cell lymphomas, especially with Sézary syndrome, and is a result of specific hypomethylation of its promoter. Since the expression of TMEM244 and the hypomethylation of its promoter are specific to T-cell lymphoma, with the highest expression in SS, they could be used as a diagnostic marker in this type of CTCL.