1: Oncogene. 2004 Oct 14;23(47):7772-9. 

Identification of an epigenetically silenced gene, RFX1, in human glioma cells
using restriction landmark genomic scanning.

Ohashi Y, Ueda M, Kawase T, Kawakami Y, Toda M.

Neuro-immunology Research Group, Keio University, School of Medicine, Tokyo,
Japan.

To identify the CpG islands differentially methylated in human glioma, we
performed restriction landmark genomic scanning with a CpG methylation-sensitive
enzyme. We found 12 spots, the intensity of which was entirely lost or decreased
in both the human glioma tissues examined as compared with that in matched
normal lymphocytes, indicating aberrant methylation of these CpG islands in
gliomas. The expression of RFX1, one of the genes associated with the methylated
CpG islands, was frequently decreased in human glioma cell lines and tissues. We
also demonstrated that the isolated CpG island located in the seventh intron of
the RFX1 gene had enhancer activity and was hypermethylated in all of the glioma
tissues and cell lines analysed, but not in normal brains or lymphocytes.
Treatment of glioma cells with a demethylating agent, 5-azacytidine, resulted in
the expression of RFX1, indicating that the silencing of the RFX1 gene may be
attributable to its methylation. RFX1 has been implicated in transcriptional
downregulation of the proto-oncogene c-myc. By expression of the RFX1 gene, the
cellular proliferative activity of glioma cells was suppressed. Taken together,
these results suggest that the RFX1 gene may be epigenetically silenced in human
gliomas and involved in glioma tumorigenesis.

PMID: 15334059 [PubMed - indexed for MEDLINE]


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2: Oncogene. 2002 May 13;21(21):3414-21. 

The proto-oncogene c-myc in hematopoietic development and leukemogenesis.

Hoffman B, Amanullah A, Shafarenko M, Liebermann DA.

Fels Institute for Cancer Research and Molecular Biology, Department of
Biochemistry, Temple University School of Medicine, 3307 North Broad Street,
Philadelphia, Pennsylvania, PA 19140, USA. hoffman@unix.temple.edu

The proto-oncogene c-myc has been shown to play a pivotal role in cell cycle
regulation, metabolism, apoptosis, differentiation, cell adhesion, and
tumorigenesis, and participates in regulating hematopoietic homeostasis. It is a
transcription regulator that is part of an extensive network of interacting
factors. Most probably, different biological responses are elicited by different
overlapping subsets of c-Myc target genes, both induced and suppressed. Results
obtained from studies employing mouse models are consistent with the need for at
least one, and possibly two, mutations in addition to deregulated c-myc for
malignant tumor formation. Repression of c-myc is required for terminal
differentiation of many cell types, including hematopoietic cells. It has been
shown that deregulated expression of c-myc in both M1 myeloid leukemic cells and
normal myeloid cells derived from murine bone marrow, not only blocked terminal
differentiation and its associated growth arrest, but also induced apoptosis,
which is dependent on the Fas/CD95 pathway. There is evidence to suggest that
the CD95/Fas death receptor pathway is an integral part of the apoptotic
response associated with the end of the normal terminal myeloid differentiation
program, and that deregulated c-myc expression can activate this signaling
pathway prematurely. The ability of egr-1 to promote terminal myeloid
differentiation when co-expressed with c-myc, and of c-fos to partially abrogate
the block imparted by deregulated c-myc on myeloid differentiation, make these
two genes candidate tumor suppressors. Several different transcription factors
have been implicated in the down-regulation of c-myc expression during
differentiation, including C/EBPalpha, CTCF, BLIMP-1, and RFX1. Alterations in
the expression and/or function of these transcription factors, or of the c-Myc
and Max interacting proteins, such as MM-1 and Mxi1, can influence the
neoplastic process. Understanding how c-Myc controls cellular phenotypes,
including the leukemic phenotype, should provide novel tools for designing drugs
to promote differentiation and/or apoptosis of leukemic cells.

Publication Types:
    Review
    Review, Tutorial

PMID: 12032779 [PubMed - indexed for MEDLINE]


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3: J Biol Chem. 2000 Oct 13;275(41):32227-33. 

Activation of protein kinase C induces nuclear translocation of RFX1 and
down-regulates c-myc via an intron 1 X box in undifferentiated leukemia HL-60
cells.

Chen L, Smith L, Johnson MR, Wang K, Diasio RB, Smith JB.

Department of Pharmacology and Toxicology and Comprehensive Cancer Center,
Schools of Medicine and Dentistry, University of Alabama at Birmingham,
Birmingham, Alabama 35294, USA.

Treatment of human promyelocytic leukemia cells (HL-60) with phorbol
12-myristate 13-acetate (PMA) is known to decrease c-myc mRNA by blocking
transcription elongation at sites near the first exon/intron border. Treatment
of HL-60 cells with either PMA or bryostatin 1, which acutely activates protein
kinase C (PKC), decreased the levels of myc mRNA and Myc protein. The inhibition
of Myc synthesis accounted for the drop in Myc protein, because PMA treatment
had no effect on Myc turnover. Treatment with PMA or bryostatin 1 increased
nuclear protein binding to MIE1, a c-myc intron 1 element that defines an
RFX1-binding X box. RFX1 antiserum supershifted MIE1-protein complexes.
Increased MIE1 binding was independent of protein synthesis and abolished by a
selective PKC inhibitor, which also prevented the effect of PMA on myc mRNA and
protein levels and Myc synthesis. PMA treatment increased RFX1 in the nuclear
fraction and decreased it in the cytosol without affecting total RFX1.
Transfection of HL-60 cells with myc reporter gene constructs showed that the
RFX1-binding X box was required for the down-regulation of reporter gene
expression by PMA. These findings suggest that nuclear translocation and binding
of RFX1 to the X box cause the down-regulation of myc expression, which follows
acute PKC activation in undifferentiated HL-60 cells.

PMID: 10918054 [PubMed - indexed for MEDLINE]


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4: FEBS Lett. 2000 May 26;474(1):23-8. 

Multiprotein complexes present at the MIF motifs flanking the promoter of the
human c-myc gene.

Itkes A, Allegra CJ, Zajac-Kaye M.

Department of Developmental Therapeutics, Medicine Branch, Division of Clinical
Sciences, National Cancer Institute, Navy Oncology Branch, Building 8, R 5101,
Naval Hospital, 20889, Bethesda, MD, USA.

The activated c-myc allele in Burkitt's lymphoma is associated with a clustering
of somatic mutations within a discrete domain of intron I that define protein
recognition sequences, designated as myc intron factors (MIF-1, MIF-2 and
MIF-3). We have previously shown that MIF-1 binding activity consists of two
polypeptides, myc intron binding polypeptide (MIBP1) and RFX1. In the present
study we identified two polypeptides, p105 and p115, and showed that these
proteins give rise to a DNA-protein complex at the MIF-2 as well as the adjacent
MIF-1 site. In addition, we demonstrated that all four proteins interact with a
novel MIF-1 like motif upstream from the c-myc promoter region, designated
5'MIF. These data suggest a model, where the interactions of MIBP1/RFX1 and
p105/p115 with the MIF-like sites may play a role in the promoter topology of
the c-myc gene.

PMID: 10828444 [PubMed - indexed for MEDLINE]


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5: Biochem J. 2000 Feb 1;345 Pt 3:535-41. 

Induction of Myc-intron-binding polypeptides MIBP1 and RFX1 during retinoic
acid-mediated differentiation of haemopoietic cells.

Zajac-Kaye M, Ben-Baruch N, Kastanos E, Kaye FJ, Allegra C.

Department of Developmental Therapeutics, Medicine Branch, Division of Clinical
Sciences, National Cancer Institute, Bethesda, MD 20889, USA.
Kayem@exchange.nih.gov

Retinoic acid-mediated differentiation of HL60 cells is associated with an
alteration of chromatin structure that maps to protein-binding sequences within
intron I of the c-myc gene and with down-regulation of c-myc expression. By
using HeLa cell extracts, we previously identified two polypeptides, designated
MIBP1 (for Myc-intron-binding peptide) and RFX1, that interact in vivo and bind
to the intron I element; we showed that tandem repeats of an MIBP1/RFX1-binding
site can exhibit silencer activity on a heterologous promoter. Here we
demonstrate that p160 MIBP1 and p130 RFX1 are absent from undifferentiated HL60
cells. In addition, we show that treatment with retinoic acid induces both MIBP1
and RFX1 protein, as well as their DNA-binding activity, upon granulocytic
differentiation of HL60 cells, with a gel mobility pattern identical to that of
HeLa cells. In the absence of p160 MIBP1 and p130 RFX1, we observed that the
altered gel mobility-shift pattern detected in undifferentiated HL60 cells
reflects the binding of two novel polypeptides, p30 and p97, that can be
cross-linked to the same recognition intron sequence. We also show that the time
course of MIBP1 and RFX1 induction is inversely correlated with the
down-regulation of c-myc levels during the treatment of HL60 cells with retinoic
acid.

PMID: 10642512 [PubMed - indexed for MEDLINE]


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6: Int J Oncol. 1999 Jul;15(1):173-8. 

Identification of c-myc promoter-binding protein and X-box binding protein 1 as
interleukin-6 target genes in human multiple myeloma cells.

Wen XY, Stewart AK, Sooknanan RR, Henderson G, Hawley TS, Reimold AM, Glimcher
LH, Baumann H, Malek LT, Hawley RG.

Oncology Research, University Health Network, Toronto, Ontario M5G 2M1, Canada.

Interleukin-6 (IL-6) is implicated in the in vivo proliferation of malignant
plasma cells in multiple myeloma. To define the molecular basis of the
IL-6-induced mitogenic response in myeloma cells, we applied STAR (subtractive
transcriptional amplification of mRNA), a new differential expression analysis
technology, to isolate mRNAs preferentially expressed in IL-6-treated versus
untreated cultures of the factor-responsive myeloma cell line U266. From the
resulting collection of STAR clones, sequence information was obtained for a
total of 72 distinct transcripts. Of these, 29 were found to correspond to known
genes, 22 matched expressed sequence tags in public databases and 21 showed no
sequence similarity to any existing entries. Among the known genes uncovered in
the screen were those encoding proteins that function in cell division, cell
signalling and gene/protein expression. Northern blot analysis documented that
two transcription factor genes chosen for further study, c-myc promoter-binding
protein (MBP-1) and X-box binding protein 1 (XBP-1), were up-regulated in U266
cells about 3-fold relative to the cell cycle-dependent beta-actin gene 12 h
after IL-6 treatment. Both genes were also similarly up-regulated by IL-6 in
factor-dependent ANBL-6 myeloma cells. These results indicate that MBP-1 and
XBP-1 are IL-6 genes in myeloma cells; as such, they may play a role in
IL-6-mediated growth control in multiple myeloma.

PMID: 10375612 [PubMed - indexed for MEDLINE]


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7: J Virol. 1996 Sep;70(9):6060-6. 

Interactions of the transcription factors MIBP1 and RFX1 with the EP element of
the hepatitis B virus enhancer.

Blake M, Niklinski J, Zajac-Kaye M.

Laboratory of Biological Chemistry, National Cancer Institute, Bethesda,
Maryland 20892, USA.

We previously demonstrated that MIBP1 and RFX1 polypeptides associate in vivo to
form a complex that binds to the MIF-1 element in the c-myc gene and the major
histocompatibility complex class II X-box recognition sequence. We now show that
the EP element, a key regulatory sequence within hepatitis B virus enhancer I,
also associates with MIBP1 and RFX1. Using polyclonal antisera directed against
either oligonucleotide-purified MIBP1 or a peptide derived from the major
histocompatibility complex class II promoter-binding protein RFX1, we showed
that MIBP1 and RFX1 are both present in the DNA-protein complexes at the EP
site. In addition, while the EP element can act cooperatively with several
adjacent elements to transactivate hepatitis B virus expression, we demonstrated
that the EP site alone can repress transcription of simian virus 40 promoter in
a position- and orientation-independent manner, suggesting a silencer function
in hepatocarcinoma cells.

PMID: 8709229 [PubMed - indexed for MEDLINE]


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8: Mol Cell Biol. 1995 Jun;15(6):3041-8. 

The myc intron-binding polypeptide associates with RFX1 in vivo and binds to the
major histocompatibility complex class II promoter region, to the hepatitis B
virus enhancer, and to regulatory regions of several distinct viral genes.

Reinhold W, Emens L, Itkes A, Blake M, Ichinose I, Zajac-Kaye M.

Laboratory of Biological Chemistry, National Cancer Institute, Bethesda,
Maryland 20892, USA.

We demonstrated that MIF-1, identified initially as a binding activity that
associated with the intron I element of the c-myc gene, consists of two
polypeptides, the myc intron-binding peptide (MIBP1) and the major
histocompatibility class II promoter-binding protein, RFX1. Using a polyclonal
antiserum directed against either oligonucleotide affinity-purified MIBP1 or a
peptide derived from RFX1, we showed that MIBP1 and RFX1 are distinct molecules
that associate in vivo and are both present in DNA-protein complexes at the
c-myc (MIF-1) and major histocompatibility complex class II (RFX1) binding
sites. We have also found that MIBP1 and RFX1 bind to a regulatory site (termed
EP) required for enhancer activity of hepatitis B virus. In addition, we have
identified MIF-1-like sequences within regulatory regions of several other viral
genes and have shown that MIBP1 binds to these sites in cytomegalovirus,
Epstein-Barr virus, and polyomavirus. We have also demonstrated that the MIF-1
and EP elements can function as silencers in the hepatocarcinoma HepG2 and the
cervical carcinoma HeLa cell lines. These findings indicate that MIBP1 and
EP/RFX1 can associate in vivo and may regulate the expression of several
distinct cellular and viral genes.

PMID: 7760800 [PubMed - indexed for MEDLINE]


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9: Nature. 1995 Apr 27;374(6525):822-3. 

Stimulation of RNA polymerase II transcription initiation by recruitment of TBP
in vivo.

Klages N, Strubin M.

Department of Genetics and Microbiology, University Medical Centre (CMU),
Geneva, Switzerland.

Eukaryotic transcriptional activators may stimulate RNA polymerase II activity
by promoting assembly of preinitiation complexes on promoters through their
interactions with one or more components of the basal machinery. On the basis of
its central role in initiating transcription-complex formation upon binding to
the TATA box, the general transcription factor TFIID, which includes the
TATA-binding protein (TBP) and several TBP-associated factors, has been
implicated as a target for activators. Consistent with this idea, an increasing
number of activators have been reported to bind directly to TBP. To assess the
functional importance of these in vitro interactions for transcriptional
regulation in vivo, we made use of a novel strategy in yeast to show that a
physical interaction with TBP is sufficient for a sequence-specific DNA-binding
protein to increase initiation of transcription by RNA polymerase II. These
results imply that binding of TFIID to promoter elements is a limiting step in
transcription complex assembly in vivo.

PMID: 7723829 [PubMed - indexed for MEDLINE]


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10: Mol Cell Biol. 1993 Nov;13(11):6810-8. 

The major histocompatibility complex class II promoter-binding protein RFX
(NF-X) is a methylated DNA-binding protein.

Zhang XY, Jabrane-Ferrat N, Asiedu CK, Samac S, Peterlin BM, Ehrlich M.

Department of Biochemistry, Tulane Medical School, New Orleans, Louisiana 70112.

A mammalian protein called RFX or NF-X binds to the X box (or X1 box) in the
promoters of a number of major histocompatibility (MHC) class II genes. In this
study, RFX was shown to have the same DNA-binding specificity as methylated
DNA-binding protein (MDBP), and its own cDNA was found to contain a binding site
for MDBP in the leader region. MDBP is a ubiquitous mammalian protein that binds
to certain DNA sequences preferentially when they are CpG methylated and to
other related sequences, like the X box, irrespective of DNA methylation. MDBP
from HeLa and Raji cells formed DNA-protein complexes with X-box
oligonucleotides that coelectrophoresed with those containing standard MDBP
sites. Furthermore, MDBP and X-box oligonucleotides cross-competed for the
formation of these DNA-protein complexes. DNA-protein complexes obtained with
MDBP sites displayed the same partial supershifting with an antiserum directed
to the N terminus of RFX seen for complexes containing an X-box oligonucleotide.
Also, the in vitro-transcribed-translated product of a recombinant RFX cDNA
bound specifically to MDBP ligands and displayed the DNA methylation-dependent
binding of MDBP. RFX therefore contains MDBP activity and thereby also EF-C, EP,
and MIF activities that are indistinguishable from MDBP and that bind to
methylation-independent sites in the transcriptional enhancers of polyomavirus
and hepatitis B virus and to an intron of c-myc.

PMID: 8413274 [PubMed - indexed for MEDLINE]


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