A. Ceribelli

B. Yao

E.K..L. Chan

P.R. Dominguez-Gutierrez

AIR Universita degli studi di Milano

Lupus T cells switched on by DNA hypomethylation via
microRNAs?
Angela Ceribelli, MD, Bing Yao, MS, Paul R. Dominguez-Gutierrez, BS, and Edward K.L.
Chan, PhD
Department of Oral Biology, University of Florida, Gainesville, FL 32610-0424, USA
The article by Zhao S et al (1) in this issue of Arthritis and Rheumatism gives further insight
into the influence of certain microRNAs (miRNAs) on the promoter methylation status
regulated by DNA methyltransferases (DNMTs). In recent years, there has been an
increased interest in the role of epigenetic modifications of DNA and pathogenetic
mechanisms of human diseases such as Systemic Lupus Erythematosus (SLE) (2).
Epigenetics is linked to stable and potentially heritable changes in gene expression that do
not entail a change in the DNA sequence. DNA methylation and histone modifications are
the two major changes that contribute to the epigenome of a cell. The first mechanism
usually occurs in the context of CpG dinucleotides at the 5' position of cytosine in the
promoter region, leading to functional consequences such as transcriptional repression. In
fact, some tissue-specific genes are silenced by promoter methylation (2). Post-translational
modifications that occur in histones make up a second group of epigenetic modifications.
Both DNA methylation and histone modifications are coupled through different
machineries, including DNMTs as well as histone modifying enzymes in multiprotein
complexes (2). Three main types of DNMTs are involved in genomic DNA methylation:
DNMT1, DNMT3A and DNMT3B. Whereas DNMT1 preferentially replicates existing
methylation patterns and maintains DNA methylation, DNMT3A and DNMT3B are
responsible for establishing new DNA methylation markers therefore referred to de novo
DNA methyltransferase (3).
A remarkable example of disease in which epigenetic-DNA methylation abnormalities and
patterns of inheritance are extremely complex is SLE, characterized by the production of a
variety of autoantibodies against nuclear and cytoplasmic components associated with
inflammation and injury of multiple organs. The high incidence of twin pairs in which only
one of the siblings has developed SLE supports the notion that environmental factors and
their involvement in epigenetic modifications could affect the onset of the disease. The
influence on SLE onset could occur at several levels. The epigenetic deregulation of genes
can contribute to, or increase, the activation of apoptosis. Moreover, it may lead to an
exacerbated activation of T and B cells (2). In fact, the DNA extracted from T cells of SLE
patients is hypomethylated compared with the DNA from normal T cells (3). The
mechanisms by which hypomethylated T cells induce SLE are not well understood.
Additional evidence of the role of global methylation changes in the development of SLE
comes from studies with DNA demethylating drugs, such as 5-azacytidine, procainamide
and hydralazine (2). In all cases, exposing T cells to demethylating drugs results in the
demethylation-dependent induction of lupus-like disease (2).
Address correspondence and reprint requests to: Edward K.L. Chan, Ph.D., Department of Oral Biology, University of Florida, 1395
Center Drive, Gainesville, FL 32610-0424. Tel: 352-273-8849. Fax: 352-273-8829. echan@ufl.edu..
Conflict of interests: The authors have no competing interests.
NIH Public Access
Author Manuscript
Arthritis Rheum. Author manuscript; available in PMC 2012 May 1.
Published in final edited form as:
Arthritis Rheum. 2011 May ; 63(5): 1177–1181. doi:10.1002/art.30192.
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MiRNAs are small, non-coding RNAs, usually 21–23 nucleotides long, which mediate post-
transcriptional silencing of target genes (4). MiRNAs usually bind to partially
complementary sites in the 3' untranslated region (UTR) of target mRNAs, and efficient
mRNA targeting requires continuous base-pairing of miRNA nucleotides 2 to 8, the so
called “seed sequence” (4). In this way, miRNAs can regulate target gene expression by
either translational inhibition, mRNA degradation or both (3,4). Dysregulation of miRNAs
by several mechanisms has been described in various disease states, including SLE (5,6).
Only recent works have suggested that miRNAs can regulate DNA methylation by targeting
the DNA methylation machinery in SLE (3) (Figure 1, panel A). The discovery of the
association between miRNAs and methylation regulation provides an insight into the role of
miRNAs in lupus CD4+ T cell hypomethylation and the pathogenesis of SLE.
Pan W et al (3) recently identified two miRNAs, miR-21 and miR-148a, to be upregulated in
CD4+ T cells in both lupus patients and MRL/lpr mice. Moreover, both miRNAs
downregulate the protein level of the enzyme DNMT1, one of the major components in the
demethylation of DNA, thus resulting in hypomethylation status in CD4+ T cells. In
particular, miR-21 indirectly downregulates DNMT1 by targeting its upstream regulator,
RASGRP1, while miR-148a directly downregulates DNMT1 by targeting the protein coding
region of its transcript (Figure 1, panel B). The final result is the derepression of
autoimmune-associated methylation-sensitive genes in CD4+T cells, such as CD70 and
LFA-1(CD11a). These investigators were also able to induce the potential alleviation of
hypomethylation in CD4+ T cells from patients with lupus by transfection with miR-21 and
miR-148a inhibitors.
The article published in this issue by Zhao S et al (1) further expands the role of miRNAs
and epigenetic changes in SLE. The novel finding is that, among 11 miRNAs found to be
increased or decreased in SLE CD4+ T cells, miR-126 was significantly overexpressed and
its upregulation was inversely correlated with DNMT1 protein levels (Figure 1, panel B).
These investigators were then able to demonstrate that miR-126 can directly inhibit DNMT1
translation by interacting with its 3'UTR region, leading to a significant reduction in the
DNMT1 protein levels (Figure 1, panel B). Through this mechanism, overexpression of
miR-126 causes demethylation and upregulation of genes encoding for LFA-1(CD11a) and
CD70, two autoimmune-related proteins, which are directly proportional to disease activity.
The inhibition of miR-126 in SLE CD4+ T cells has opposite effects. The miR-126 host
gene EGFL7 was also overexpressed in SLE CD4+ T cells, in a hypomethylation-dependent
manner.
Another interesting point of the work by Zhao S et al (1) is that these investigators focused
their attention not only in the influence of the DNA methylation machinery on lupus CD4+
T cells, but also on the co-stimulation between active T cells and B cells, leading to IgG
overproduction. They were also able to show that knocking-down miR-126 in SLE CD4+ T
cells reduced their autoimmune activity and their stimulatory effect on IgG production in the
co-cultured B cells.
The main differences in the reports by Pan et al (3) and Zhao et al (1) focusing on the role of
miRNA overexpression affecting the DNA methylation mechanism in SLE T cells are
shown in Table 1. Pan W et al (3) and Zhao et al (1) detected different miRNAs involved in
the DNA methylation machinery. One possible explanation is that Pan W et al (3) performed
the microarray analysis of miRNAs in splenic CD4+ T cells and B cells isolated from MRL/
lpr mice and normal control mice, but not in samples from SLE patients. On the contrary, in
the present study, Zhao S et al (1) perform this analysis from CD4+ T cells of SLE patients
and healthy controls. However, even though the initial microarray analyses were performed
on different substrates, both of them proceed with the miRNA analysis on SLE patients, as
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shown in Table 1. Moreover, it is well known that SLE is a very heterogeneous disease, so it
is possible that the different altered miRNAs detected in the two works can be associated
with the selection of patients, disease subsets, autoantibody expression, immunosuppressive
therapy, and phases of disease activity. Ethnic background is usually another important
aspect to consider when studying SLE patients, but it does not seem to play a role in this
case because both groups of SLE patients are from Chinese population.
One possible limitation in the work by Zhao S et al (1) is the lack of information on SLE
patients, in terms of clinical features and immunosuppressive therapy. Patients were
recruited from in-patient ward and dermatology department, and it is not clear whether these
SLE patients had predominantly dermatological manifestations, which could be a reason for
the different miRNA expression in the two studies. Moreover, Pan W et al (3) provide more
detailed information on SLE patients, including disease duration, anti-dsDNA titer, number
of patients affected by lupus nephritis, detection of proteinuria, and use of
immunosuppressive therapy.
Both studies analyze the influence of DNA methylation on the expression of genes that are
linked to T cell autoreactivity. The CD70 and LFA-1(CD11a) genes are the targets studied
in both reports, as they are demethylated genes overexpressed post-treatment with
hypomethylating agents in CD4+ T cells (3). Zhao S et al (1) further studied a new target,
the miR-126 host gene EGFL7, which is also overexpressed in SLE CD4+ T cells. MiR-126
upregulation is associated with a reduction of EGFL7 promoter methylation and thus also
with T cell autoreactivity in SLE, leading to an amplification cycle that further contributes to
the disease.
Zhao S et al (1) study the regulation of miR-126 on DNMT1 at the 3' UTR level through the
use of miRBase information. However, this prediction was not confirmed by other
algorithms, such as TargetScan or PicTar, commonly used in these bioinformatic analyses.
This may be particularly important because, in the present work, the 5' interaction of
miR-126 and the 3' UTR of DNMT1 only involve 4 bases. It is generally known that the
seed sequence is expected to be 7–8 bases for most strong miRNA-mRNA interactions.
Thus, it could be possible that the proposed interaction is weak and the in vivo biological
relevance needs to be further examined (Figure 1, panel A).
It is also clear that miRNAs can regulate the immune response through pathways
independent from the DNA methylation machinery (7). In fact, recent reports have shown
that miR-146a is a negative regulator of the Interferon (IFN) pathway in lupus patients, and
that underexpression of miR-125 contributes to the elevated expression of the pro-
inflammatory cytokine RANTES in lupus (5–7).
A number of analytical techniques are now available for studying epigenetic modifications
in a genome-wide manner. The systematic use of these genomic techniques will serve to
provide a full profile of epigenetic deregulation in SLE. Unlike genetic alterations, which
are permanent, epigenetic alterations are reversible. This opens the possibility of using
epigenetic drugs to reverse the pattern of epigenetic alterations to relieve the phenotype. To
date, histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid
(SAHA) and trichostatin A (TSA) have proved to be useful for relieving SLE disease in
mice (2). The effects of TSA on human T cells are predominantly immunosuppressive and
reminiscent of the signaling aberrations that have been described in patients with SLE (2).
Current evidence indicates that most of the genes that exhibit aberrant patterns of DNA
methylation are hypomethylated, although gene-gene-specific hypermethylation cannot be
ruled out (2). Therefore, the detailed analysis of DNA methylation at the gene level will
serve to evaluate how useful histone deacetylase inhibitors and DNA demethylating drugs
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could be. It is not clear whether the increased expression of specific miRNAs is an indirect
effect rather than the cause of SLE, and also this point needs further investigation in future
studies.
Acknowledgments
This work was supported in part by a grant from the Lupus Research Institute and the National Institutes of Health
grant AI47859. P.R.D.G. was supported by NIH training grant T32 DE007200.
REFERENCES
1. Zhao S, Wang Y, Liang Y, Zhao M, Long H, Ding S, et al. MicroRNA-126 regulates DNA
methylation in CD4+ T cells and contributes to systemic lupus erythematosus by targeting DNA
methyltransferase 1. Arthritis Rheum. 2010
2. Ballestar E, Esteller M, Richardson BC. The epigenetic face of systemic lupus erythematosus. J
Immunol. 2006; 176:7143–7. [PubMed: 16751355]
3. Pan W, Zhu S, Yuan M, Cui H, Wang L, Luo X, et al. MicroRNA-21 and microRNA-148a
contribute to DNA hypomethylation in lupus CD4+ T cells by directly and indirectly targeting DNA
methyltransferase 1. J Immunol. 2010; 184:6773–81. [PubMed: 20483747]
4. Krol J, Loedige I, Filipowicz W. The widespread regulation of microRNA biogenesis, function and
decay. Nat Rev Genet. 2010; 11:597–610. [PubMed: 20661255]
5. Tang Y, Luo X, Cui H, Ni X, Yuan M, Guo Y, et al. MicroRNA-146A contributes to abnormal
activation of the type I interferon pathway in human lupus by targeting the key signaling proteins.
Arthritis Rheum. 2009; 60:1065–75. [PubMed: 19333922]
6. Zhao X, Tang Y, Qu B, Cui H, Wang S, Wang L, et al. MicroRNA-125a contributes to elevated
inflammatory chemokine RANTES via targeting KLF13 in systemic lupus erythematosus. Arthritis
Rheum. 2010
7. Pauley KM, Cha S, Chan EK. MicroRNA in autoimmunity and autoimmune diseases. J Autoimmun.
2009; 32:189–94. [PubMed: 19303254]
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FIGURE 1.
MiR-21, miR-126, and miR-148a directly or indirectly regulate DNMT1 in SLE CD4+ T
cells.
A. MiR-148a and miR-126 directly regulate DNMT1 validated experimentally. In particular,
miR-148a binds to the DNMT1 coding sequence, as predicted by the program RNA22,
while miR-126 binds to the 3'-UTR region of DNMT1, as predicted by the program
Microcosm.
B. DNMT1 is directly or indirectly regulated in SLE CD4+ T cells and it triggers the over-
expression of autoimmune-associated methylation-sensitive genes involved in SLE. In
normal human T cells, gene hypermethylation controlled by DNMT1 blocks the
transcription activity of RNA pol II, thus silencing autoimmune-related methylation-
sensitive genes such as CD70, LFA-1(CD11a) and EGFL7. In SLE CD4+ T cells, promoter
hypomethylation allows binding of transcriptional factors (TF) and recruiting of RNA pol II
(step 1). This leads to the overexpression of autoimmune-related genes, including EGFL7
(step 2). Interestingly, miR-126 is encoded in the 7th intron of EGFL7. Thus the expression
of EGFL7 leads to elevated levels of miR-126, which downregulates the production of
DNMT1 in a reciprocal negative feedback (amplification) loop (step 3). Low level of
DNMT1 leads to the hypomethylation status which maintains the aberrant autoimmune
response (step 4). Other miRNAs contributing to decreased DNMT1 activity are miR-148a,
through direct inhibition, and miR-21 which indirectly regulates DNMT1, by targeting
RASGRP1 in the Ras-MAPK pathway, and thus also influencing DNMT1 protein levels.
DNMT1=DNA methyltransferase 1; TF= transcription factor; RNA pol II= RNA
polymerase II; RASGRP1= RAS guanyl-releasing protein 1; bp= base pairs.
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TABLE 1
Main differences in the two recent reports focusing on the role of miRNA overexpression affecting the DNA
methylation mechanism in SLE T cells.
Pan W et al (3) Zhao S et al (1)
Overexpressed miRNAs MiR-21, miR-148a MiR-126
Cell substrate used for microarray
analysis
Splenic CD4+ T cells and B cells isolated from
MRL/lpr mice and controls
CD4+ T cells of SLE patients and healthy
controls, no mouse model
Number of SLE patients studied for
miRNA expression
36 (33 female, 3 male) 30 (all female)
SLE patients' information Complete (male/female ratio, age, disease
duration, SLEDAI score, anti-dsDNA, lupus
nephritis, use of steroids and secondary agents)
Limited (age, gender, SLEDAI score,
medications)
Overexpressed methylation-sensitive
gene targets
LFA-1(CD11a), CD70 LFA-1(CD11a), CD70, EGFL7
Cell types examined for miRNA
overexpression
CD4+ T cells CD4+ T cells and B cells
Arthritis Rheum. Author manuscript; available in PMC 2012 May 1.


Lupus T cells switched on by DNA hypomethylation via microRNA?

https://air.unimi.it/retrieve/handle/2434/251847/344354/2011_Lupus%20T%20cells%20switched%20on%20by%20DNA%20hypomethylation%20via%20microRNA.pdf

Lupus T cells switched on by DNA hypomethylation via microRNA?

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