KLF feedback loops in innate immunity

Abstract

The Krüppel-like factor (KLF) family of zinc finger transcription factors regulate the expression of genes involved in a wide range of cellular processes, including cell proliferation and differentiation. In haematopoiesis, KLFs have essential roles in myeloid cell differentiation and function. KLF4 is a critical regulator of macrophage development and initiates pro- and anti-inflammatory signalling pathways in response to various stimuli. KLF2, KLF3 and KLF6 also play important roles in regulating these pathways. Here we review how KLFs cooperate and compete to either activate or repress target genes to influence initiation and resolution of inflammatory responses in macrophages. We also discuss how KLFs may be involved in the development of chronic inflammatory conditions.

Keywords: KLF3; KLF4; chronic inflammatory disease; feedback loops; inflammation; innate immunity; macrophage; transcriptional regulation.

Figure 1

Biochemistry of the KLF family. (A) Schematic of murine KLF4, KLF3, KLF2, KLF1 and KLF6. The DNA-binding domain of three C2H2 zinc fingers is at the very C-terminus. The N-terminus contains well defined domains and motifs that recruit different cofactors such as p300/CBP, CtBPs, and others. (B) The DNA binding domain of all SP/KLFs binds an 8-9bp motif in vitro and in vivo. This model is a composite derived from the structures of SP1 and KLF4 bound to different dsDNA molecules (5, 6). Each nucleotide is color-coded. Redundancy of binding specificity at positions 7 and 5 on the G-rich strand is indicated by colour grading. Position 9 on the G-rich strand is white to reflect redundancy for any nucleotide for the KLF4 clade but specificity for a G for SP factors and some KLFs. The key amino acids in ZF1, ZF2 and ZF3 (relative to an alpha helix) of murine KLF4 and the nucleotides they contact are indicated. (C) Group 2 KLFs (KLF1/2/4/5/6/7) recruit p300/CBP and associated proteins to acetylate chromatin tails (H3K27ac) and well as lysines in KLF4. They recruit SMARCA4 and associated SWI/SNF proteins to open chromatin and pioneer for other transcription factors. Group 1 KLFs (KLF3/8/12) recruit CtBP1/2 and subsequently HDACs to deacetylate chromatin, as well as complexes containing LSD1 and Co-REST. (D) KLF4 post-translational modifications are critical for responses to external signals and effector functions. ERK1/2 phosphorylate KLF4, E3 ubiquitin ligases induce lysine ubiquitination and subsequent degradation and PRMT1/5 induce arginine dim-methylation which has functional consequences. (E) KLFs bind cis regulator modules in enhancers and promoters with a specific syntax with respect to other transcription factors such as PU.1, CEBPA, IRFs, and REL/NF-κB to co-ordinate gene expression in macrophages and other cell types. These motifs are found in different combinations within KLF4-regulated enhancers and promoters of macrophage genes.

Figure 2

Coherent and Incoherent Feed Forwards Loops regulate transcription of target genes in a dynamic tuneable fashion. (A) KLFs can work in coherent feed-forward networks to amplify gene expression (e.g. KLF2/4/5 in ES cell pluripotency maintenance) (44) (B) KLFs can work in incoherent feed-forward loops to initiate a transient gene expression program followed by silencing after a period of time (20, 45). (C) There is redundancy in negative regulation of KLF networks. KLF3 can repress KLF8 and KLF12, which are themselves negative regulators of the upstream positive and negative regulators of target gene expression (45).

Figure 3

Expression of many KLF/SP family members in monocytes and macrophages. (A) Data mining from the Hemosphere online RNA-seq database derived from (51). SP1 is ubiquitously expressed in all blood cell lineages. (B–F) KLFs 2, 3, 4 and 6 are all highly expressed in FACS-sorted monocytes and macrophages from the blood and bone marrow. On the other hand, KLF8 is expressed at low levels in all blood types, KLF3 is also high in erythroid cells whereas KLF4, KLF2 and KLF6 is not. (G) KLF1 is highly expressed in erythroid cell but not in monocytes.

Figure 4

Monocyte/macrophage differentiation and KLF expression in monocytes and tissue-resident macrophages. (A) The stages of macrophage development. Yolk-sac erythro-myeloid progenitors differentiate into primitive yolk-sac derived macrophages that are long-lived. Foetal liver and bone marrow derived hematopoietic stem cells (HSCs) give rise to short-lived and long-lived macrophages during development and adulthood. A combination of these different macrophage sources contribute to the populations of adult tissue-resident macrophages (B–D) Data mining for a suite of different GEO submissions of different macrophage datasets for KLF expression. This bioinformatic analysis of these diverse datasets was undertaken by (53). It is difficult to compare absolute expression levels of different KLFs but relative expression of KLF2, KLF4 and KLF2 shows marked differences. For example, KLF3 is relatively highly expressed in alveolar macrophages and gut-derived macrophages.

Figure 5

Macrophage differentiation and M1/M2 polarisation. A Summary of KLF influences on monocyte differentiation and macrophage polarisation. Monocytes differentiate from common myeloid progenitors (CMP) and granulocyte-macrophage progenitors (GMP), where KLF4 favours monocyte over neutrophil lineages. Monocytes then migrate to tissues as naïve M0-type macrophages. Upon inflammatory stimulus they polarise to M1-type pro-inflammatory or M2-anti-inflammatory macrophages. The influences of KLFs on the different stages of differentiation and polarisation are summarised.

Figure 6

The KLF3 -TLR loci in human and mouse. (A) Schematic generated from the UCSC Genome Browser (GRCh38/hg38). The KLF3 gene sits just upstream and is expressed in the opposite direction to three TLR genes, TLR10, TLR1 and TLR6, and sits downstream of the TBC1D1 gene within a 1Mb region of DNA on chromosome 4p14. There are a large number of lncRNAs (green) and enhancer signatures in this region between the coding genes, including KLF3-AS (121). There are three SNPs in the vicinity of one of these spliced LNC RNAs that are associated with inflammatory bowel disease (119, 120). The entire region is syntenic with mouse chr5qC3.1. (B) Schematic generated from the UCSC Genome Browser (CRCm38/mm10). The Klf3 gene sits just upstream and is expressed in the opposite direction to two TLR genes, Tlr1 and Tlr6, and sits downstream of the Tbc1d1 gene within a 1Mb region of DNA on chromosome 5qC3.1.