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40 Part I Molecular and Cellular Basis of Hematology

increase transcription, while phosphorylation marks damaged DNA. transcript elongation can occur. RNA polymerase then continues to
Histone methylation can either open chromatin to increase transcrip- traverse the template DNA strand, using ATP while complementarily
tion, or close it to repress transcription, depending on where the pairing bases and forming the phosphodiester–ribose backbone.
histone is methylated. Ubiquitination is the enzymatic addition or Many RNA transcripts may be rapidly produced from a single copy
removal of the ubiquitin moiety from histones. Transcription factors of a gene, as multiple RNA polymerases may be transcribing the gene
can themselves recruit histone-modifying enzymes that can regulate simultaneously, spaced out from one another. An important proof-
transcription. In hematopoiesis, transcription factors including reading mechanism during elongation allows the substitution of
GATA-1, ELKF, NF-E2 and PU.1 recruit histone acetyltransferases incorrectly incorporated bases, usually by permitting short pauses
(HATs) and histone deacetylases (HDACs) to promoters of target during which the appropriate RNA editing factors can bind. RNA
genes, leading to addition or subtraction of acetyl groups from his- editing mechanisms in mRNAs include nucleoside modifications of
tones, thereby affecting chromatin structure and the openness of cytidine to uridine (C-U) and adenosine to inosine (A-I) by deamina-
DNA to transcription. A gene essential to erythroid maturation and tion, as well as nucleotide insertions and additions without a DNA
survival, GATA-1, for instance, directly recruits HAT complexes to template by proteins called editosomes.
the β-globin locus to stimulate transcription activation. Another repair mechanism is transcription-coupled nucleotide
Chromatin usually tightly packages DNA, which is essential for excision repair, where RNA polymerase stops transcribing when it
the cell to have a functional size and shape. Therefore, for transcrip- comes to a bulky lesion in one of the nucleotides in the gene. A large
tion to take place, the DNA must be unwound from the chromatin. protein complex excises the DNA segment containing the bulky
This process of unpackaging is called chromatin remodeling and is lesion, and a new DNA segment is synthesized to replace it, using
mediated by a family of proteins with switch/sucrose nonfermentable the opposite strand as a template. The RNA polymerase then resumes
SWI/SNF domains. These proteins use ATP hydrolysis to shift the transcribing the gene. However, in general, RNA proofreading
nucleosome core along the length of the DNA, a process also known mechanisms are not as effective as in DNA replication, and transcrip-
as nucleosome sliding. By sliding nucleosomes away from a gene tion fidelity is lower.
sequence, SWI/SNF complexes can activate gene transcription. After a gene is transcribed, mRNA is modified to protect it and
SWI/SNF proteins also contain helicase enzyme activity, which target it for translation to protein. These modifications include
unwinds the DNA by breaking hydrogen bonds between the comple- capping and polyadenylation. Capping occurs shortly after the start
mentary nucleotides on opposite strands. By unwinding the DNA of transcription, when a modified guanine nucleotide is added to the
into two single strands, the DNA can then be read by RNA poly- 5′ end of the mRNA. This terminal 7-methylguanosine residue is
merases in the direction 3′ to 5′. A new antiparallel RNA strand, 5′ necessary for proper attachment to the ribosome during translation.
to 3′, is produced by RNA polymerases to mirror the coding strand It also protects the RNA from endogenous ribonucleases that degrade
of the DNA, with the exception of all thymine nucleotides replaced uncapped RNA, which is often viral in origin.
by uracil nucleotides. SWI/SNF proteins have the ability to utilize RNA polymerases do not terminate transcription in an orderly
Brahma (BRM) or Brahma-related gene 1 (BRG1) as alternative cata- manner. They tend to be processive, yet the cell cannot tolerate a
lytic subunits with ATPase activity to remodel chromatin. The SWI/ population of mRNAs that are enormous in size. Therefore, mRNAs
SNF complex has been shown to be active in the DNA damage have a signal, the sequence AAUAA, that defines the end of the
response and is also responsible for tumor suppression. More recently, transcript. Ribonucleases cut mRNAs shortly after that signal, and a
BRM and BRG1 have been proposed as independent tumor suppres- chain of several hundred adenosine residues is added to that free 3′
sors; however, their role in hematologic malignancies is not known. transcript end. Synthesis of this poly(A) tail and termination of
DNA itself can be chemically modified to amplify or suppress transcription requires binding of specific proteins, including cleavage/
transcription. CpG sites with gene promoter regions can be chemi- polyadenylation specificity factor (CPSF), cleavage stimulation factor
cally modified by methylation enzymes DNA methyltransferases (CstF), polyadenylate polymerase (PAP), polyadenylate binding
(DNMTs), which subsequently decrease binding of RNA polymerase protein 2 (PAB2), cleavage factor I (CFI), and cleavage factor II
and associated transcription factors. Hypermethylation has been (CFII), that function to catalyze cleavage and protect the mRNA
observed in bone marrow cells of patients with myelodysplastic from exoribonucleases. The poly(A) tail also assists in export of the
syndromes (MDS) and the degree of DNA hypermethylation cor- mRNA from the nucleus and translation. Mutations in the poly(A)
relates with disease stage. In MDS the promoters of genes that are signal can result in hematologic disease. For example, there are
important for myeloid differentiation are hypermethylated, repress- thrombophilic patients with a mutation in the polyadenylation signal
ing their transcription, and inhibiting proper maturation of the in the prothrombin gene that increases the stabilization of this
myeloid lineages. Hypomethylating agents such as azacitidine and mRNA, resulting in higher prothrombin protein levels and increased
decitabine can induce remission and prolonged survival in MDS thrombosis.
patients. The regulation of gene expression by modification of chro-
matin or DNA itself is termed epigenetic, as it alters cell function
without altering the nucleotide sequence of the DNA. RNA SPLICING
Such epigenetic modifications are crucial to the behavior of
hematologic diseases. Mutation of the DNMT3 genes may have Before the mRNA can be translated into protein, introns must be
indirect effects on gene expression without altered DNA methylation, removed and the exons re-connected (Fig. 4.4). This process, termed
as have been observed in 20% of acute myeloid leukemia (AML) cases splicing, requires a series of reactions mediated by the spliceosome,
and are correlated with poor clinical outcome. The Ten-Eleven- a complex of small nuclear ribonucleoproteins (snRNPs). The types
Translocation oncogene member, TET2, which plays a role in DNA of snRNPs in the spliceosome determine the mechanism of splicing.
methylation and therefore epigenetic stability, is mutated in AML, Canonical splicing, also called the lariat pathway, utilizes the major
MDS, chronic myelomonocytic leukemia (CMML), and other spliceosome and accounts for more than 99% of splicing. The major
myeloproliferative neoplasms (MPNs). Another recurring observa- spliceosome is composed of the nuclear active snRNPs U1, U2, U4,
tion in blood malignancies is aberrant histone methylation, for U5, and U6 along with specific accessory proteins, U2AF and SF1.
example at H3K27, seen in myelodysplasia. This is associated with This complex recognizes the dinucleotide GU at the 5′ end of an
altered gene expression affecting cell cycle, cell death, and cell adhe- intron, and an AG at the 3′ end. Intermediately a lariat structure
sion pathways. forms, connecting these ends, providing for both excision of the
Before a final mRNA product is made that can be translated, intron and proper alignment of the ends of the two bordering exons
several proofreading regulatory steps must take place. The RNA to allow precise ligation. When the intronic flanking sequences do
polymerase may not even clear the promoter and slip off, producing not follow the GU-AG rule, noncanonical splicing removes these rare
truncated transcripts. Once the transcript reaches approximately introns with different splice site sequences using the minor spliceo-
23 nucleotides, the RNA polymerase no longer slips off, and full some. The same U5 snRNP is found in the minor spliceosome, in