PU.1 and partners: regulation of haematopoietic stem cell fate in normal and malignant haematopoiesis

Abstract

During normal haematopoiesis, cell development and differentiation programs are accomplished by switching 'on' and 'off' specific set of genes. Specificity of gene expression is primarily achieved by combinatorial control, i.e. through physical and functional interactions among several transcription factors that form sequence-specific multiprotein complexes on regulatory regions (gene promoters and enhancers). Such combinatorial gene switches permit flexibility of regulation and allow numerous developmental decisions to be taken with a limited number of regulators. The haematopoietic-specific Ets family transcription factor PU.1 regulates many lymphoid- and myeloid-specific gene promoters and enhancers by interacting with multiple proteins during haematopoietic development. Such protein-protein interactions regulate DNA binding, subcellular localization, target gene selection and transcriptional activity of PU.1 itself in response to diverse signals including cytokines, growth factors, antigen and cellular stresses. Specific domains of PU.1 interact with many protein motifs such as bHLH, bZipper, zinc fingers and paired domain for regulating its activity. This review focuses on important protein-protein interactions of PU.1 that play a crucial role in regulation of normal as well as malignant haematopoiesis. Precise delineation of PU.1 protein-partner interacting interface may provide an improved insight of the molecular mechanisms underlying haematopoietic stem cell fate regulation. Its interactions with some proteins could be targeted to modulate the aberrant signalling pathways for reversing the malignant phenotype and to control the generation of specific haematopoietic progeny for treatment of haematopoietic disorders.

Figure 1

Functional domains of PU.1 protein along with their interacting proteins. The N-terminus Transactivation domain interacts with TFIID, TBP, GATA-1, GATA-2, CBP and retinoblastoma protein. The C-terminus ETS domain codes for a DNA-binding domain that recognizes the sequence 5′-GGAA-3′. The ETS domain interacts with c-Jun, c-Myb, GATA-1, GATA-2, C/EBP-α, NFIL6-β, AML-1b, AML-ETO, etc. The PEST domain interacts with PIP (PU.1 interacting partner) and ICSBP. The phosphorylation at Ser148 is essential for this interaction.

Figure 2

PU.1 interacts through its c-terminal ETS domain with the leucine zipper domain of NF-IL6β. Both the proteins simultaneously bind to DNA during complex formation and the transcriptional synergy results from each protein independently influencing components of basal transcriptional complex.

Figure 3

c-Jun acts as an important co-activator of transcription factor PU.1 during the gene regulation of various myeloid gene promoters such as M-CSF receptor promoter and macrosialin promoter. c-Jun physically interacts with the β₃/β₄ region in the ETS domain of PU.1 and functionally activates it. Generally, c-Fos heterodimerizes with c-Jun but it blocks the coactivation of PU.1 by c-Jun.

Figure 4

CBP/p300 functions by bridging between sequence-specific transcriptional activators and general transcription factors of the basal transcription machinery. CBP/p300 enhances transcription by targeted acetylation of specific chromatin domains with their intrinsic histone acetyltransferase activities. The region of CBP spanning residues 1283–1915 interacts with a portion of the TAD of PU.1 (aa residues 74–122) directly and acts as its co-activator. CBP also binds and stimulates the activity of erythroid-specific transcription factor GATA-1 by acetylating its two highly conserved lysine-rich motifs near each of the two zinc fingers.

Figure 5

Cross-antagonism between PU.1 and GATA-1. GATA-1 represses PU.1 function by interacting through its c-terminal zinc finger with the β₃/β₄ region of PU.1 and displacing its co activator c-Jun. On the other hand, PU.1 represses GATA-1 function by interacting through its transactivation domain with the c-terminal zinc finger of GATA-1 and thereby inhibiting its binding to the cognate DNA sequence.

Figure 6

The activation of neutrophil elastase gene is regulated by the cooperative interaction between c-Myb, C/EBP-α and PU.1 through their respective DNA binding domains.

Figure 7

The M-CSF receptor gene is regulated by transcription factor PU.1 in combination with C/EBP-α and AML-1B. PU.1, C/EBP-α and AML-1B interact through their respective DNA binding domains and transactivation domains. AML-1 exhibits cooperative DNA binding with C/EBP-α but not with PU.1. The co-activator CBF-β serves to amplify the activation by increasing transcription efficiency.

Figure 8

The fusion protein AML-1/ETO inhibits the function of PU.1 by displacing its co-activator c-Jun from its β₃/β₄ region.

Figure 9

PU.1 recruits PIP (PU.1 interacting partner) by binding with its regulatory domain through its PEST domain. For PU.1’s interaction with Pip, phosphorylation of Ser148 residue is essential. RD, regulatory domain; AD, activation domain; DBD, DNA binding domain.

Figure 10

PU.1-protein interactions play a vital role in HSC fate determination. Co-activator c-Jun interacts with the β₃/β₄ region of PU.1 and enhances its transcriptional ability that in turn leads to increased formation of monocyte/macrophages. When the relative concentration of GATA-1 is higher, it inhibits the PU.1-c-Jun interaction by displacing c-Jun from its binding site and thus blocking the formation of monocytes. The up-regulated activity of GATA-1 in turn leads to enhanced erythropoiesis. Similarly, transcription factor C/EBP-α also inhibits PU.1-c-Jun interaction and leads to the formation of granulocytes. Additionally, a fusion protein AML-1/ETO also represses PU.1 by inhibiting its interaction with co-activator c-Jun, leading to maturation arrest of myeloblasts and thus causing myeloid leukaemia.